Note: Descriptions are shown in the official language in which they were submitted.
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DELIVERY OF OPHTHALMOLOGIC AGENTS
TO THE EXTERIOR OR INTERIOR OF THE EYE
FIELD OF THE INVENTION
100011 The invention relates generally to drug-eluting polymer compositions
and in particular
to biodegradable, biocompatible polymer delivery compositions for ocular
delivery of
ophthalmologic agents in a controlled time release fashion.
BACKGROUND INFORMATION
100021 Many implantable drug delivery devices have been developed over the
past several
years. Such drug delivery devices may be formulated from synthetic or natural,
biodegradable
or non-biodegradable, polymers. Biodegradable polymers are preferred since
these materials
gradually degrade in vivo over time, e.g., by enzymatic or non-enzymatic
hydrolysis, when
placed in an aqueous, physiological environment. Thus, the use of
biodegradable polymers in
drug delivery devices is preferred since their use avoids the necessary
removal of the drug
delivery device at the end of the drug release period. =
100031 The drug is generally incorporated into the polymeric composition
and formed into
the desired shape outside the body. This solid implant is then typically
inserted into the body of
a human, animal, bird, and the like through an incision. Alternatively, small
discrete particles
composed of these polymers can be injected into the body by a syringe.
100041 Certain of these polymers also can be injected via syringe as a
liquid polymeric
composition. These compositions are administered to the body in a liquid state
or, alternatively,
as a solution. Once in the body, the composition coagulates into a solid. One
type of polymeric
composition includes a nonreactive thermoplastic polymer or copolymer
dissolved in a body
fluid-dispersible solvent. This polymeric solution is placed into the body
where the polymer
congeals or precipitatively solidifies upon dissipation or diffusion of the
solvent into the
surrounding body tissues.
100051 In particular, nonbiodegradable polymer implants, most commonly
various types of
methylmethacrylate, have been used for local delivery of antibiotics, such as
Tobramycin,
gentamicin, and vancomycin. A biodegradable antibiotic implant made of
polylactic acid and
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poly(DL-lactide): co-glycolide combined with vancomycin has been developed and
evaluated in
a localized Osteomyelitic rabbit model (Cahoun JH et al. Clinical Orthopaedics
& Related
Research. Current Trends in the Management of Disorders of the Joints. (1997)
341:206-214).
100061 More recently the GIADEDID wafer (Guilford Pharmaceutical Corp,
Baltimore, MD),
which was FDA-approved for implant in post surgical treatment of certain kinds
of brain tumors,
is used to deliver an oncolytic agent, carmustine, from a wafer of a
biodegradable polyanhydride
copolymer. Hydrolytic degradation products of Gliadel wafer (in addition to
the anticancer
agent) are ultimately the starting di-acids: sebacic acid and I,3-bis(4-
carboxyphenoxy) propane
(CPP). Clinical investigations of Gliadel implants in rabbit brains have shown
limited toxicity.
initial activitiand fast excretion of decomposition products - the free acids
(A.J. Domb et al.
Biomaterials. (1995) 16:1069-1072).
100071 Local drug delivery from implants provides the advantage of high
tissue
concentrations with relatively low serum levels, thereby avoiding some of the
toxicity associated
with systemic delivery. Bioactive agent-impregnated polymer implants are
particularly
attractive because they not only deliver high tissue levels of antibiotic or
oncolytic agent, but
also help fill the dead space that occurs after certain surgeries. However,
drug release
characteristics of such implanted drug delivery devices may be suboptimal.
Many times, the
release of pharmacologically active agents from an implanted drug delivery
device is irregular.
There is an initial burst period when the drug is released primarily from the
surface of the
device, followed by a second period during which little or no drug is
released, and a third period
during which most of the remainder of the drug is released at a substantially
lower rate than in
the initial burst.
100081 More recently CPP was disclosed as a monomer useful in preparation
of
bioabsorbable stents for vascular applications by "Advanced Cardiovascular
Systems, Inc", in
-patent WO 03/080147 Al, 2003 and polymer particles in U.S. provisional
application
Serial No. 60/684,670, filed May 25, 2005.
[00091 Another aromatic biodegradable di-acid monomer based on.trans-4-
hydroxycirmamic
acid has been recently described. The monomer with general name 4,4'-
(alkanedioyldioxy)
dicinnamic acid inherently contains two hydrolytically labile ester groups,
and is expected to
undergo specific (enzymatic) and nonspecific (chemical) hydrolysis (M Nagata,
Y. Sato.
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Polymer. (2004) 45:87-93). The biodegradable polymers containing unsaturated
groups have
potential for various applications. For example, unsaturated groups can be
converted into other
functional groups such as epoxy or alcohol ¨ useful for further modifications.
Their crosslinlcing
could enhance thermal stability and mechanical properties of polymer.
Cinnamate is known to
undergo reversible [2 + 2] cyclo-addition upon UV irradiation at wavelengths
over 290 nm,
without presence of photoinitiator, a property which makes the polymer self-
photo-crosslinkable
(Y. Nakayama, T. Matsuda. J. Polym. Sci. Part A: Polym. Chem. (1992) 30:2451-
2457). In
addition, the cinnamoyl esters are metabolized in the body and have been
proven to be non-toxic
(Citations in paper of M Nagata, Y. Sato. Polymer. (2004) 45:87-93).
100101 Recent research has also shown that hydrogel-type materials can be
used to shepherd
various medications through the stomach and into the more alkaline intestine.
Hydrogels are
cross-linked, hydrophilic, three-dimensional polymer networks that are highly
permeable to
various drug compounds, can withstand acidic environments, and can be tailored
to "swell" and
thereby release entrapped molecules through their weblike surfaces. Depending
on the chemical
composition of the gel, different internal and external stimuli (e.g., changes
in pH, application of
a magnetic or electric field, variations in temperature, and ultrasound
irradiation) may be used to
trigger the swelling effect. Once triggered, however, the rate of entrapped
drug release is
determined solely by the cross-linking ratio of the polymer network.
10011] Delivery of drugs intraocularly is a particular problem. The eye is
divided into two
chambers; the anterior segment which is the front of the eye, and the
posterior segment which is
the back of the eye. Diseases of the anterior segment are easier to treat with
formulations such
as eye drops because they can be applied topically. For example, glaucoma can
be treated from
the front of the eye. Diseases of the retina, such as diabetic retinopathy and
macular
degeneration, are located in the posterior segment and are difficult to treat
because drugs applied
topically, such as eye drops, typically do not penetrate to the back of the
eye. Drugs for these
disease states have customarily been delivered by injection directly into the
back of the eye.
100121 Researchers have sought to overcome these difficulties. The topical
administration of
a cationic emulsion onto the eye has been shown to increase the residence time
of a lipophilic
drug on the cornea, with a lower contact angle and an increased spreading
coefficient in
comparison with conventional eye drops and anionic emulsions. In the case of
the posterior
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segment of the eye, certain cationic emulsions for non-invasive topical
administration have been
developed that allow a lipophilic drug to migrate to the retina via the trans-
scleral route from the
cornea and conjunctiva, which act as a reservoir.
[00131 Chemists, biochemists, and chemical engineers are all looking beyond
traditional
polymeric and other formulations to find innovative drug transport systems.
Thus, there is still a
need in the art for new and better polymer delivery compositions for
controlled delivery of a
variety of different types of bioactive agents to target specific body sites,
such as the exterior and
interior tissues of the eye. In particular, there is a need in the art for new
and better polymer
delivery compositions for continuous delivery of an ophthalmologic agent to
the anterior or
posterior segment of the eye over a sustained period of time, for example in
treatment of chronic
diseases of the front and back of the eye.
SUMMARY OF THE INVENTION
100141 The present invention is based on the premise that polymers
containing at least one
amino acid and a moiety that is not an amino acid per repeat unit, such as
polyester amide (PEA)
polyester urethane (PEUR) and polyester urea (PEU) polymers, can be used to
formulate
biodegradable polymer delivery compositions for time release of ophthalmologic
agents in a
consistent and reliable manner into the exterior or interior of the eye by
biodegradation of the
polymer.
100151 The present invention is also based on the premise that PEAs, PEURs
and PEUs can
be formulated as polymer delivery compositions that incorporate a therapeutic
agent (e.g., a
residue of an ophthalmologic diol) into the backbone of the polymer for time
release into the
exterior or interior of the eye in a consistent and reliable manner by
biodegradation of the
polymer. The invention intraocular polymer delivery composition may optionally
also deliver
into the exterior or interior of the eye another type of bioactive agent that
is dispersed in the
polymer.
100161 In one embodiment, the invention provides an intraocular polymer
delivery
composition comprising at least one ophthalmologic agent dispersed in at least
one
biodegradable polymer, wherein the composition is implantable in the exterior
or interior of the
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eye and the polymer comprises at least one of a PEA having a chemical formula
described by
structural formula (I),
49 9 9 OH
R3 R3 A
Formula (1)
wherein n ranges from about 5 to about 1 50; RI is independently selected from
residues of a,w-
allcylene dicarboxylates of formula (III) below, or in combination with (C2 -
Cm) alkylene, (C2-
C20) alkenylene, am-bis(4-carboxyphenoxy)-(C1-C8) alkane, 3,3 '-
(alkanedioyldioxy) dicinnamic
acid or 4,4'-(alkanedioyldioxy) dicinnamic acid, or saturated or unsaturated
residues of
therapeutic di-acids; and wherein R6 and R6 in formula (III) are independently
selected from (C2
- C12) alkylene or (C2-C12) alkenylene; the R3s in individual n units are
independently selected
from the group consisting of hydrogen, (Ci-C6) alkyl, (C2-C6) alkenyl, (C2-C8)
allcynyl, (C6-C10)
aryl (Ci-C6) alkyl, and -(CH2)2S(CH3); and R4 is independently selected from
the group
consisting of (C2-C20) alkylene, (C2-C20) alkenylene, (C2-C8) allcyloxy (C2-
C20) allcylene,
bicyclic-fragments of 1,4:3,6-dianhydrohexitols of structural formula (II),
saturated or
unsaturated therapeutic diol residues, and combinations thereof;
CH
\
H2C/ ICH2
\o = /
CH
Formula (II)
0 0 0 0
HO-C-R6-C-0-R6-0-6-0-6-0H =
Formula (111)
or a PEA polymer having a chemical formula described by structural formula
(IV):
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-
0 0 0 0 H
" --111-811--9 9 ____
C-O-R4-0-C-cili-H 6-111-6-N-6¨R13-Nii
{ 1
R H
m I I 2 I
H 9-0-R H p n
0
Formula (1V)
wherein n ranges from about 5 to about 150, m ranges about b. i to 0.9; p
ranges from about 0.9
to O. l; RI is independently selected from residues of am-alkylene
dicarboxylates of structural
formula (111), or in combination with (C2 - Cu) alkylene and (C2-C20)
alkenylene, a,co-bis(4-
carboxyphenoxy)-(Ci-CB) alkane, 3,3'-(alkanedioyldioxy) dicinnamic acid,
4,4'-(alkanedioyldioxy)dicinnamic acid, saturated or unsaturated residues of
therapeutic di-acids
and combinations thereof; and wherein R5 and R6 in Formula (III) are
independently selected
from (C2 - Ci2) alkylene or (C2-C12) alkenylene; each R2 is independently
hydrogen, (C1-C12)
alkyl, (C6-C10) aryl or a protecting group; the les in individual m monomers
are independently
selected from the group consisting of hydrogen, (C1-C6) alkyl, (C2-C6)
alkenyl, (C2-C6) allcynyl,
(C6-C1o) aryl (C1-C6) alkyl, and -(CH2)2S(CH3); and R4 is independently
selected from the group
consisting of (C2-C20) alkylene, (C2-C20) alkenylene, (C2-C8) alkyloxy (C2-
C20) alkylene,
bicyclic-fragments of 1,4:3,6-dianhydrohexitols of structural formula (II),
residues of saturated
or unsaturated therapeutic diols, and combinations thereof: and R13 is
independently (C1-C2o)
alkyl or (C2-C20) alkenyl.
[00171 In still another embodiment, the invention provides
methods for delivering an
ophthalmologic agent to the exterior or interior of the eye of a subject by
administering to the
subject ocularly an invention intraocular polymer delivery composition
comprising one or more
polymers of structural formulas I and IV-VIII and at least one ophthalmologic
agent dispersed in
said one or more polymers, which composition biodegrades by enzymatic action
to release the
ophthalmologic agent(s) to the exterior or interior of the eye at a controlled
rate.
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[0017a] Further embodiments of the invention include:
- an intraocular polymer delivery composition comprising an ophthalmologic
agent dispersed in a biodegradable polymer, wherein the polymer comprises a
poly(ester
amide) (PEA) having formula (I):
OH
_________________________________________________________ R1 -N C C -R4 -O E -
N
11 iIV _
Formula (I), wherein n ranges from 5 to 150; R1 is independently selected from
residues of
a,w-bis(4-carboxyphenoxy)(Ci-C8)alkane, 3,3'-(alkanedioyldioxy)dicinnamic
acid, 4,4'-
(alkanedioyldioxy)dicinnamic acid, (C2-C20)alkylene, (C2-C20)alkenylene,
residues of a,co-
alkylene dicarboxylates of formula (III), and combinations thereof;
0 o 0 0
HO-C¨R"-C-0¨R6-0-8-R5-8-0H
Formula (III), wherein R5 and R6 in Formula (III) are each independently
selected from
(C2-C12)alkylene and (C2-C12)alkenylene; the R3s in individual n monomers are
each
independently selected from hydrogen, (Ci-C6)alkyl, (C2-C6)alkenyl,
(C6-Cio)aryl(C i-C6)alkyl, and ¨(CH2)2S(CH3); and R4 is independently selected
from
(C2-C20)alkylene, (C2-C20) alkenylene, (C2-C8)a1ky1oxy, (C2-C20)alkylene,
bicyclic-fragments
of 1,4:3,6-dianhydrohexitols of formula (II), and combinations thereof:
CH
CH2
0 CH
Formula (II), or a PEA having a chemical formula described by structural
formula (IV):
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6b
, 9 ki 9 OH 9
t
{ 2 t 1
ig --12*-C1¨?¨C-0-R4-0-C13 1;1 C R1 u rC¨R1311
H R3 R H H C-O-R2 H
m P n
0
Formula (IV), wherein n ranges from 5 to 150, m ranges 0.1 to 0.9, p ranges
from 0.9 to 0.1;
Ri is independently selected from residues of am-bis (4-carboxyphenoxy)(Ci-
C8)alkane,
3,3'-(alkanedioyldioxy)dicinnamic acid, 4,4'-(alkanedioyldioxy)dicinnamic
acid, residues of
a,a)-alkylene dicarboxylates of formula (III), (C2-C2o)alkylene, (C2-
C2o)alkenylene and
combinations thereof wherein; R5 and R6 in Formula (III) are each
independently selected
from (C2-C12)alkylene and (C2-Ci2)alkenylene; R2 is independently selected
from hydrogen,
(CI-Ci2)alkyl, (C2-C8)alkyloxy, (C2-C20) alkyl, (C6-Cio)aryl, and a protecting
group; the R3s in
individual n monomers are each independently selected from hydrogen, (Ci-
C6)alkyl,
(C2-C6)alkenyl, (C6-Cio)ary1(Ci-C6)a1ky1 and ¨(CH2)2S(CH3); and R4 is
independently
selected from (C2-C20)alkylene, (C2-C20)alkenylene, (C2-C8)alkyloxy, (C2-
C20)alkylene,
bicyclic-fragments of 1,4:3,6-dianhydrohexitols of formula (II), and
combinations thereof;
and R13 is independently (Ci-C20)alkyl or (C2-C20) alkenyl; and
- use of the composition as described herein for delivering the ophthalmologic
agent comprised therein to the interior or exterior of an eye of a subject,
thereby releasing for
the ophthalmologic agent in a controlled manner.
DETAILED DESCRIPTION OF THE INVENTION
[0018] The invention is based on the discovery that biodegradable
polymers can be used
to create a polymer delivery composition for ocular delivery of opthalmologic
agents dispersed
within. The ocular polymer delivery compositions can be fashioned as
dispersions of particles,
as implantable solids, or as films for extra- or intraocular delivery. The
invention ocular
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polymer delivery compositions biodegrade by enzymatic hydrolytic actions so as
to release the
ophthalmologic agent at a controlled rate. The invention particle compositions
are stable, and
can be lyophilized for transportation and storage and be redispersed for
administration. Due to
structural properties of the polymer used, the invention intraocular polymer
delivery
compositions provide for high loading of ophthalmologic agents.
[0019] Accordingly, in one embodiment, the invention provides an
intraocular polymer
delivery composition comprising at least one ophthalmologic agent dispersed in
at least one
biodegradable polymer, wherein the polymer is a PEA having a chemical formula
described by
structural formula (I),
CC-R10 HO OH 1
-e-H¨C-8-0-R4-0-8-6-N
-[-
ili 43 43 ii n
Formula (I)
wherein n ranges from about 5 to about 150; RI is independently selected from
residues of a,w-
bis (4-carboxyphenoxy) (CI-C8) alkane, 3,3'-(alkanedioyldioxy) dicinnamic acid
or 4,4'-
(alkanedioyldioxy) dicinnamic acid, residues of am-alkylene dicarboxylates of
formula (III), (C2
- C20) alkylene, (C2-C20) alkenylene or a saturated or unsaturated residues of
therapeutic di-acids
and combinations thereof; and wherein R5 and R6 in Formula (III) are
independently selected
from (C2 - C12) alkylene or (C2-C12) alkenylene; the R3s in individual n
monomers are
independently selected from the group consisting of hydrogen, (C1-C6) alkyl,
(C2-C6) alkenyl,
(C2-C6) alkynyl, (C6-Cio) aryl (CI-C6) alkyl and -(CH2)2S(CH3); and R4 is
independently
selected from the group consisting of (C2-C20) alkylene, (C2-C20) alkenylene,
(C2-C8) alkyloxy
(C2-C20) alkylene, bicyclic-fragments of 1,4:3,6-dianhydrohexitols of
structural formula (II), and
combinations thereof, (C2 - C20) alkylene, (C2-C20) alkenylene, and saturated
or unsaturated
therapeutic di-acid residues;
\
H2C/ I \C H2
\ /
0 CH
\
Formula (II)
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0 0 0 0
HO-C-R"-C-0-1r-O-C-12"-C-OH
Formula (III)
or a PEA polymer having a chemical formula described by structural formula
(IV),
0 H 0
IIo
/ OIL ti 9., cl. 1-.1
CC-R'-Cii-C-C-o-R4-o-u-?-1 _________________ c-R'-i.;--rc¨R13111
H R3 R3 H H m C-0-R2 Fit, .
ii
Formula (IV)
wherein n ranges from about 5 to about 150, m ranges about 0.1 to 0.9: p
ranges from about 0.9
to 0.1; wherein R1 is independently selected from residues of a,co-bis (4-
carboxyphenoxy) (Cr
C8) alkane, 3,3 '-(alkanedioyldioxy)dicinnamic acid or 4,4'-
(alkanedioyldioxy)dicinnamic acid,
residues of a,w-alkylene dicarboxylates of formula (III) above, (C2 - Cm)
alkylene, (C2-C20)
alkenylene or a saturated or unsaturated residues of therapeutic di-acids and
combinations
thereof; wherein R5 and R6 in Formula (III) are independently selected from
(C2 - C12) alkylene
or (C2-C12) alkenylene; each R2 is independently hydrogen, (CI-C12) alkyl or
(C6-Cio) aryl or a
protecting group; the R3s in individual m monomers are independently selected
from the group
consisting of hydrogen, (C1-C6) alkyl, (C2-C6) alkenyl, (C2-C6) alkynyl, (C6-
C10) aryl (Ci-C6)
alkyl, and -(CH2)2S(CH3); and R4 is independently selected from the group
consisting of (C2-
C20) alkylene, (C2-C20) alkenylene, (C2-C8) alkyloxy (C2-C20) alkylene,
bicyclic-fragments of
1,4:3,6-dianhydrohexitols of structural formula (II), and combinations
thereof, and residues of
saturated or unsaturated therapeutic diols; and R13 is independently (C,-C20)
alkyl or (C2-C20)
alkenyl, for example, (C3-C6) alkyl or (C3-C6) alkenyl.
[0020j For example, an effective amount of the residue of at least one
therapeutic diol or di-
acid can be contained in the polymer backbone. For example, the therapeutic
diol can be an
ophthalmologic agent, as disclosed herein. Alternatively, in the PEA polymer,
at least one R1 is
a residue of a,w-bis (4-carboxyphenoxy) (C1-C8) alkane or 4,4'-
(alkanedioyldioxy) dicinnamic
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acid and R4 is a bicyclic-fragment of a 1,4:3,6-dianhydrohexitol of general
formula OD, or a
residue of a saturated or unsaturated therapeutic diol. In another
alternative, RI in the PEA
polymer is either a residue of a,(0-bis (4-carboxyphenoxy) (C1-C8) alkane, or
4,4'-(alkanedioyl
dioxy) dicinnamic acid, a residue of a therapeutic diacid, and mixtures
thereof. In yet another
alternative, in the PEA polymer RI is a residue a,co-bis (4-carboxyphenoxy)
(C1-C8) alkane, such
as 1 ,3-bis(4-carboxyphenoxy)propane (CPP), or 4,4'-(adipoyldioxy) dicinnamic
acid and R4 is a
bicyclic-fragment of a 1,4:3,6-dianhydrohexitol of general formula (II), such
as 1,4:3,6-
dianhydrosorbitol (DAS).
100211 Alternatively, the invention intraocular polymer delivery
composition can comprise at
least one ophthalmologic agent dispersed in a biodegradable polymer, wherein
the polymer
comprises a PEUR polymer having a chemical formula described by structural
formula (V),
[O 0 R6_0_g-N-161 g-O-R4-0-2-14I-Nh
HR3 R3 H
Formula (V)
and wherein n ranges from about 5 to about 150; wherein the R3s within an
individual n
monomer are independently selected from the group consisting of hydrogen, (Ci-
C6) alkyl, (C2-
C6) alkenyl, (C2-C6) alkynyl, (C6-C10) aryl(Ci-C6) alkyl and ¨(CH2)2S(CH3); R4
and R6are
independently selected from (C2-C20) alkylene, (C2-C20) alkenylene or (C2-C8)
alkyloxy (C2-C2o)
alkylene, bicyclic-fragments of 1,4:3,6-dianhydrohexitols of general formula
(II), a residue of a
saturated or unsaturated therapeutic diol, and mixtures thereof.
or a PEUR polymer having a chemical structure described by general structural
formula (VI),
0 F;I 0 OH 0 4? Fil
4:1C?-0 R6 0 a-i.,4 9 6-0 R4 0 C C ni c-o-R6-0--C-HN-Cc--0-7:231 p
{
H R3 R3 H
m 1
II
0 n
Formula (VI)
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wherein n ranges from about 5 to about 150, m ranges about 0.1 to about 0.9: p
ranges from
about 0.9 to about 0.1; R2 is independently selected from hydrogen, (C6-
Cio)aryl (Ci-C6) alkyl,
or a protecting group; the R3s within an individual m monomer are
independently selected from
the group consisting of hydrogen, (Ci-C6) alkyl, (C2-C6) alkenyl, (C2-C6)
alkynyl, (C6-Cio)
aryl(Ci-C6) alkyl, and ¨(CH2)2S(CH3); R4 and R6 are independently selected
from (C2-C2o)
alkylene, (C2-C20) alkenylene or (C2-C8) alkyloxy (C2-C20) alkylene, bicyclic-
fragments of
1,4:3,6-dianhydrohexitols of structural formula (II), a residue of a saturated
or unsaturated
therapeutic diol, and mixtures thereof; and R13 is independently (CI-CAI)
alkyl or (C2-C20)
alkenyl, for example, (C3-C6) alkyl or (C3-C6) alkenyl.
100221 For example, an effective amount of the residue of at least one
therapeutic diol,
including, but not limited to, an ophthalmologic diol, as disclosed herein,
can be contained in the
polymer backbone. In one alternative in the PEUR polymer, at least one of R4
or R6 is a bicyclic
fragment of .1,4:3,6-dianhydrohexitol, such as 1,4:3,6-dianhydrosorbitol
(DAS).
100231 In still another embodiment the invention intraocular polymer
delivery composition
can comprise at least one ophthalmologic agent dispersed in a biodegradable
polymer, wherein
the polymer comprises at least one biodegradable PEU polymer having a chemical
formula
described by structural formula (VII),
49 ki 9 OH
A R3 R3AL
Formula (VII),
wherein n is about 10 to about 150; the R3s within an individual n monomer are
independently
selected from hydrogen, (C,-C6) alkyl, (C2-C6) alkenyl, (C2-C6) alkynyl, (C6 -
Cio) aryl (Cr
C6)alkyl, ¨(CH2)3, and ¨(CH2)2S(CH3); R4 is independently selected from (C2-
C20) alkylene, (C2-
C20) alkenylene, (C2-C8) alkyloxy (C2-C20) alkylene, a residue of a saturated
or unsaturated
therapeutic diol; or a bicyclic-fragment of a 1,4:3,6-dianhydrohexitol of
structural formula (II);
or a PEU having a chemical formula described by structural formula (VIII),
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H 0 0 1;1 1 [ O I:1
21i 3 6-0 R4 0 C c3 i'i C tii-----R13-141,1
[
H c-O-R2 H
m
is PIn
0
Formula (VI11)
wherein m is about 0.1 to about 1.0; p is about 0.9 to about 0.1; n is about
10 to about 150; each
R2 is independently hydrogen, (Ci-C12) alkyl or (C6-Cio) aryl; and the R3s
within an individual m
monomer are independently selected from hydrogen, (CI-C6) alkyl, (C2-C6)
alkenyl, (C2-C6)
alkynyl, (C6 -CO aryl (Ci-C6)alkyl, and ¨(CH2)2S(CH3); R4 is independently
selected from (C2-
C2o) alkylene, (C2-C20) alkenylene, (C2-C8) alkyloxy (C2-C20) alkylene, a
residue of a saturated
or unsaturated therapeutic diol; or a bicyclic-fragment of a 1,4:3,6-
dianhydrohexitol of structural
formula (II), or a mixture thereof; and R13 is independently (C1-C20) alkyl or
(C2-C20) alkenyl,
for example, (C3-C6) alkyl or (C3-C6) alkenyl.
[0024] For example, an effective amount of the residue of at least one
therapeutic diol,
including but not limited to an ophthalmologic diol, as disclosed herein, can
be contained in the
polymer backbone. In one alternative in the PEU polymer, at least one R4 is a
residue of a
saturated or unsaturated therapeutic diol, or a bicyclic fragment of a 1,4:3,6-
dianhydrohexitol,
such as DAS. In yet another alternative in the PEU polymer, at least one R4 is
a bicyclic
fragment of a 1,4:3,6-dianhydrohexitol, such as DAS.
[0025] These PEU polymers can be fabricated as high molecular weight
polymers useful for
making polymer particles suitable for delivery into the interior or exterior
of the eye of humans
and other mammals of a variety of ophthalmologic agents. These PEUs
incorporate
hydrolytically cleavable ester groups and non-toxic, naturally occurring
monomers that contain
a-amino acids in the polymer chains. The ultimate biodegradation products of
PEUs will be
amino acids, diols, and CO2. In contrast to the PEAs and PEURs, the invention
PEUs are
crystalline or semi-crystalline and possess advantageous mechanical, chemical
and
biodegradation properties that allow formulation of completely synthetic, and
hence easy to
produce, crystalline and semi-crystalline polymer particles, for example
nanoparticles.
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10026i For example, the PEU polymers used in the invention intraocular
polymer particle
delivery compositions have high mechanical strength, and surface erosion of
the PEU polymers
can be catalyzed by enzymes present in physiological conditions, such as
hydrolases.
10027B As used herein, the terms "amino acid" and "a-amino acid" mean a
chemical
compound containing an amino group, a carboxyl group and a pendent R group,
such as the R3
groups defined herein. As used herein, the term "biological a-amino acid"
means the amino
acid(s) used in synthesis are selected from phenylalanine, leucine, glycine,
alanine, valine,
isoleucine, methionine, or a mixture thereof. As used herein, the term
"adirectional amino acid"
means a chemical moiety within the polymer chain obtained from an a-amino
acid, such that the
R group (for example R13 in formulas VI and VII) is inserted within the
polymer backbone.
10028B As used herein, a "therapeutic diol" means any diol molecule,
whether synthetically
produced, or naturally occurring (e.g., endogenously), that affects a
biological process in a
mammalian individual, such as a human, in a therapeutic or palliative manner
when
administered to the mammal.
=
100291 The PEA, PEUR and PEU polymers used in the invention compositions
are poly-
condensates. The ratios "m" and "p" in Formulas (IV, VI and VIII) are defined
as irrational
numbers in the description of these poly-condensate polymers. Moreover, as "m"
and "p" will
each take up a range within any poly-condensate, such a range cannot be
defined by a pair of
integers. Each polymer chain is a string of monomer residues linked together
by the rule that all
bis-amino acid diol (i) and adirectional amino acid (e.g. lysine) (ii) monomer
residues are linked
either to themselves or to each other by a diacid monomer residue (iii) for
PEA, by a diol residue
(iii) for PEUR or carbonyl for PEU (iii). Thus, only linear combinations of i-
iii-i; i-iii-ii (or ii-
iii-i) and ii-iii-ii are formed. In turn, each of these combinations is linked
either to themselves or
to each other by a diacid monomer residue (iii) for PEA or a diol residue
(iii) for PEUR or
carbonyl for PEU (iii). Each polymer chain is therefore a statistical, but non-
random, string of
monomer residues. Each individual polymer chain is composed of integer numbers
of
monomers, i, ii and iii. However, in general for polymer chains of any
practical average
molecular weight (i.e., sufficient mean length), the ratios of monomer
residues "m" and "p" in
formulas (IV, VI and VIII) will not be whole numbers (rational integers).
Furthermore, for the
condensate of all poly-dispersed polymer chains the numbers of monomers i, ii
and iii averaged
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over all of the chains (i.e. normalized to the average chain length)_will not
be integers. It follows
that the ratios can only take irrational values (i.e., any real number that is
not a rational number).
Irrational numbers, as the term is used herein, are derived from ratios that
are not of the form n/j,
where n and j are integers.
100301 As used herein, the term "residue of a therapeutic diol" means a
portion of a
therapeutic diol, as described herein, which portion excludes the two hydroxyl
groups of the
diol. As used herein, the term "residue of a therapeutic di-acid" means a
portion of a therapeutic
di-acid, as described herein, which portion excludes the two carboxyl groups
of the di-acid. The
corresponding therapeutic diol or di-acid containing the "residue" thereof is
used in synthesis of
the polymer compositions. The ophthalmologic agents disclosed herein are a
subset of the
therapeutic diols disclosed herein. The residue of the therapeutic di-acid or
diol is reconstituted
in vivo (or under similar conditions of pH, aqueous media, and the like) to
the corresponding di-
acid or diol upon release from the backbone of the polymer by biodegradation
in a controlled
manner that depends upon the properties of the PEA, PEUR or PEU polymer(s)
selected to
fabricate the composition, which properties are as known in the art and as
described herein.
100311 As used herein the term "bioactive agent" means a bioactive agent as
disclosed herein
that is not incorporated into the polymer backbone. One or more such bioactive
agents
optionally may be dispersed in the invention intraocular polymer delivery
compositions. As
used herein, the term "dispersed" means that the bioactive agent is dispersed,
mixed, dissolved,
homogenized, and/or covalently bound to ("dispersed") in a polymer, for
example attached to a
functional group in the biodegradable polymer of the composition or to the
surface of a polymer
particle, but not incorporated into the backbone of a PEA, PEUR, or PEU
polymer. To
distinguish backbone-incorporated therapeutic diols and di-acids from those
that are not
incorporated into the polymer backbone, (as a residue thereof), such dispersed
therapeutic or
palliative agents are referred to herein as "bioactive agent(s)" and may be
contained within
polymer conjugates or otherwise dispersed in the polymer particle composition,
as described
below. Such bioactive agents may include, without limitation, small molecule
drugs, peptides,
proteins, DNA, cDNA, RNA, sugars, lipids and whole cells. In one embodiment,
the invention
intraocular polymer delivery composition administers the ophthalmologic agent,
with or without
an optional bioactive agent dispersed therein in polymer particles having a
variety of sizes and
structures suitable to meet differing therapeutic goals and routes of
administration.
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[0032] The term, "biodegradable, biocompatible" as used herein to describe
the PEA, PEUR
and PEU polymers, including mixtures and blends thereof, used in fabrication
of invention
intraocular polymer delivery compositions means the polymer is capable of
being broken down
into innocuous products in the normal functioning of the body. This is
particularly true when the
amino acids used in fabrication of the polymers are biological L-a-amino
acids. A
"biodegradable polymer" as the term is used herein also means the polymer is
degraded by water
and/or by enzymes found in tissues of mammalian patients, such as humans. The
invention
intraocular polymer delivery compositions are also suitable as implants for
use in veterinary
treatment of a variety of mammalian patients, such as pets (for example, cats,
dogs, rabbits,
ferrets), farm animals (for example, swine, horses, mules, dairy and meat
cattle) and race horses
when used as described herein.
[0033] The term "controlled" as used herein to described the release of
bioactive agent(s)
from invention intraocular polymer delivery compositions means the polymer
implant degrades
over a desired period of time to provide a smooth and regular (i.e.
"controlled") time release
profile (e.g., avoiding an initial irregular spike in drug release and
providing instead a
substantially smooth rate of change of release throughout biodegradation of
the invention
composition).
[0034] The polymers in the invention intraocular polymer delivery
compositions include
hydrolyzable ester and enzymatically cleavable amide linkages that provide
biodegradability,
and are typically chain terminated, predominantly with amino groups.
Optionally, the amino
termini of the polymers can be acetylated or otherwise capped by conjugation
to any other acid-
containing, biocompatible molecule, to include without restriction organic
acids, bioinactive
biologics, and bioactive agents as described herein. In one embodiment, the
entire polymer
composition, and any particles made thereof, is substantially biodegradable.
[0035] In one alternative, at least one of the a-amino acids used in
fabrication of the
polymers used in the invention compositions and methods is a biological a-
amino acid. For
example, when the R3s are CH2Ph, the biological a-amino acid used in synthesis
is L-
phenylalanine. In alternatives wherein the R3s are CH2¨CH(CH3)2, the polymer
contains the
biological a-amino acid, L-leucine. By varying the R3s within monomers as
described herein,
other biological a-amino acids can also be used, e.g., glycine (when the R3s
are H), alanine
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(when the R3s are CH3), valine (when the R3s are CH(CH3)2), isoleucine (when
the R3s are
CH(CH3)¨CH2¨CH3), phenylalanine (when the R3s are CH2¨C6H5), or methionine
(when the R3s
are -(CH2)2SCH3), and mixtures thereof. In yet another alternative embodiment,
all of the
various a-amino acids contained in the polymers used in making the invention
polymer particle
or solid intraocular delivery compositions are biological a-amino acids, as
described herein.
100361 The term, "biodegradable" as used herein to describe the polymers
used in the
invention intraocular polymer delivery composition means the polymer is
capable of being
broken down into innocuous and bioactive products in the normal functioning of
the body. In
one embodiment, the entire polymer particle delivery composition is
biodegradable. The
biodegradable polymers described herein have hydrolyzable ester and
enzymatically cleavable
amide linkages that provide the biodegradability, and are typically chain
terminated
predominantly with amino groups. Optionally, these amino termini can be
acetylated or
otherwise capped by conjugation to any other acid-containing, biocompatible
molecule, to
include without restriction organic acids, bioinactive biologics and bioactive
agents.
100371 In one embodiment the invention intraocular polymer delivery
compositions are
fabricated as particles, which can be formulated to provide a variety of
properties. For example,
the polymer particles can be sized to agglomerate intraocularly, forming a
time-release polymer
depot for local delivery of dispersed ophthalmologic agents to surrounding
tissue/cells when
injected or surgically implanted therein. For example, polymer particles of
sizes capable of
passing through pharmaceutical syringe needles ranging in size from about 19
to about 27 gauge,
for example those having an average diameter in the range from about liam to
about 200 pm,
can be injected intraocularly, and will agglomerate to form particles of
increased size that form
the depot to dispense the ophthalmologic agent(s) locally.
(00381 The biodegradable polymers used* in the invention intraocular
polymer delivery
composition can be designed to tailor the rate of biodegradation of the
polymer to result in
continuous delivery of the ophthalmologic agent, with or without an additional
bioactive agent,
over a selected period of time. For instance, typically, a polymer depot, as
described herein, will
biodegrade over a time selected from about twenty-four hours, about seven
days, about thirty
days, or about ninety days, or longer, for example up to three years. Longer
time spans are
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particularly suitable for providing a delivery composition that eliminates the
need to repeatedly
inject the composition to obtain a suitable therapeutic or palliative
response.
(0039) The invention utilizes biodegradable polymer particle- or solid-
mediated delivery
techniques to deliver ophthalmologic agents into the interior or exterior of
the eye. For example
the invention compositions can be placed subconjunctivally (under the thin
membrane that
covers the top of the front of the eye except for the comea) but on top of the
sclera (white part of
the eye) to provide a sustained delivery. Alternatively, the invention
composition can be placed
surgically, as described herein, to delivery the ophthalmologic agent to the
back of the eye.
Alternatively still, the invention composition can be placed underneath the
Tenon's capsule on
top of the sclera to allow diffusion of the ophthalmologic agent through the
sclera from the back
of the eye to the retina. This mode of emplacement is called "subtenon"
delivery. Thus, the
invention compositions can be used to deliver ophthalmologic agents in
treatment of a wide
variety of ophthalmologic diseases and disease symptoms.
[00401 As used herein, the terms "intraocular", "intraocularly" and "into
the interior of the
eye" mean subconjunctival, transcleral, or subtenon delivery of an active
agent, but not topical
delivery.
[00411 Although certain of the individual components of the polymer
particle and solid
delivery compositions and methods described herein were known, it was
unexpected and
surprising that such combinations would enhance the efficiency of time release
delivery of the
ophthalmologic agents beyond levels achieved when the components were used
separately.
100421 The PEA, PEUR and PEU polymers used in practice of the invention
bear
functionalities that allow facile covalent attachment to the polymer of the
ophthalmologic agent
and, optionally, other bioactive agent(s) or covering molecule(s). For
example, a polymer
bearing carboxyl groups can readily react with an amino moiety, thereby
covalently bonding a
peptide to the polymer via the resulting amide group. As will be described
herein, the
biodegradable polymer and the bioactive agent may contain numerous
complementary functional
groups that can be used to covalently attach an ophthalmologic or other
bioactive agent to the
biodegradable polymer. Alternatively, such polymers used in the invention
compositions, such
as the polymer particles, and methods are ready for reaction with other
chemicals having a
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17
hydrophilic structure to increase water solubility and with covering
molecules, without the
necessity of prior modification.
[0043] In addition, the polymers disclosed herein (e.g., those having
structural formulas (I
and IV-VIM, upon enzymatic degradation, provide amino acids while the other
breakdown
products can be metabolized in the way that fatty acids and sugars are
metabolized. Uptake of
the polymer with bioactive agent is safe: studies have shown that the subject
can
metabolize/clear the polymer degradation products. These polymers and the
invention
intraocular polymer delivery compositions are, therefore, substantially non-
inflammatory to the
subject both at the site of injection and systemically, apart from the trauma
caused by injection
itself.
[0044] The biodegradable PEA, PEUR and PEU polymers useful in forming the
invention
biocompatible intraocular polymer particle and solid delivery compositions may
contain multiple
different a-amino acids in a single polymer molecule, for example, at least
two different amino
acids per repeat unit, or a single polymer molecule may contain multiple
different a-amino acids
in the polymer molecule, depending upon the size of the molecule. The polymer
may also be a
block co-polymer. In another embodiment, the polymer is used as one block in
di- or tri-block
copolymers, which are used to make micelles, as described below.
100451 In addition, the polymers used in the invention polymer particle and
solid delivery
compositions display minimal hydrolytic degradation when tested in a saline
(PBS) medium, but
in an enzymatic solution, such as chymotrypsin or CT, a uniform erosive
behavior has been
observed.
[0046] Suitable protecting groups for use in the PEA, PEUR and PEU polymers
include t-
butyl or another as is known in the art. Suitable 1,4:3,6-dianhydrohexitols of
general formula
(11) include those derived from sugar alcohols, such as D-glucitol, D-
mannitol, or L-iditol.
Dianhydrosorbitol is the presently preferred bicyclic fragment of a 1,4:3,6-
dianhydrohexitol for
use in the PEA, PEUR and PEU polymers used in fabrication of the invention
polymer particle
delivery compositions.
100471 The PEA, PEUR and PEU polymer molecules may also have an ophthalmologic
or
other bioactive agent attached thereto, optionally via a linker or
incorporated into a crosslinker
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between molecules. For example, in one embodiment, the polymer is contained in
a polymer-
bioactive agent conjugate having structural formula (IX):
{ZO o H (?! 01.4
.-
CRI- -N-264 0 R4 OUC N R14 N CH (CH2)4 NH
123 1
R3 in
c=o PIn
R5
R7
Formula (IX)
wherein n, m, p, RI, R3, and R4 are as above, R5 is selected from the group
consisting of¨O-, -S-
and -NR8-, wherein R8 is H or (CI-C8)alkyl; and R7 is the bioactive agent.
100481 In yet another embodiment, two molecules of the polymer of
structural formula (X)
can be crosslinked to provide an -R5-R7-R5- conjugate. In another embodiment,
as shown in
structural formula (IX) below, the bioactive agent is covalently linked to two
parts of a single
polymer molecule of structural formula (IV) through the -R5-R7-R5- conjugate
and R5 is
independently selected from the group consisting of¨O-, -S-, and -NR8-,
wherein R8 is H or (C1-
C8) alkyl; and R7 is the ophthalmologic or other bioactive agent.
R3 R3 \
H 1 H H
C-Ftl--N-C¨(CH2)4-NH _____________________________________________________
IS H 11 11
0 11
0
0
R7 ______ R5 0
R5 ________________________________
R3 R3
= ______________________________ (C H2)4 C -NH-C-R1-C ____________________ l
fNH-C-C-O-R4-O-C-CH-N-C-R1-C
11 11 l t H " fl 11 11
l
0 0
0 0 0 n
Formula (X)
100491 Alternatively still, as shown in structural formula (XI) below, a
linker, can be
inserted between R5 and bioactive agent R7, in the molecule of structural
formula (IV), wherein
X is selected from the group consisting of (CI-Cis) alkylene, substituted
alkylene, (C3-C8)
cycloalkylene, substituted cycloalkylene, 5-6 membered heterocyclic system
containing 1-3
heteroatoms selected from the group 0, N, and S, substituted heterocyclic, (C2-
C18) alkenyl,
substituted alkenyl, alkynyl, substituted alkynyl, C6 and Cio aryl,
substituted aryl, heteroaryl,
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substituted heteroaryl, allcylaryl, substituted alkylaryl, arylalkynyl,
substituted arylalkynyl,
arylalkenyl, substituted arylalkenyl, arylalkynyl, substituted arylalkynyl and
wherein the
substituents are selected from the group H, F, CI, Br, I, (C1-C6) alkyl, -CN, -
NO2, ¨OH, -0(C1-
C4) alkyl, -S(C1-C6) alkyl, -SR=0)(C1-C6) alkyl], -S[(02)(Ci-C6) alkyl], -
C[(=0)(Ci-C6) alkyl],
CF3,-ORCO)-( CI-C6) alkyl], -S(02)[N(R9RIN, -MIRC'0)(Ci-C6) alkyl],
-NH(C=0)N(R9RIG), -N(R9RI ); where R9 and RI are independently H or (Ct-C6)
alkyl; and Y
is selected from the group consisting of¨O-, -S-, -S-S-, -S(0)-,-S(02)-, -NR8-
, -C(=0)-, -
OC(=0)-, -C(=0)0-, -0C(=0)NH-, -NR8C(=0)-, -C(=0)NR8-, -N R8C(=0)NR8-,
-N R8C(=0)NR8-, and -NR8C(=S)N R8-.
/
c) c) 00
io It H H li 92 H H
-
, 9 H
C-R1-C-N---C-0-R4-0 C C N ______________ C R'-C N CH (CH2)4 NH
-
R3 RI3 M l
{
C=0 P}n
i
R5
X
I
Y
1
R7
Formula (XI)
[00501 In another embodiment, two parts of a single macromolecule are
covalently linked to
the ophthalmologic or other bioactive agent through an ¨R5-R7-Y-X- R5- bridge
(Formula XII):
R3 R3
H I i H H
-R1-9-N-C.--O-R4-0--CH-N C-R1--N-C¨(CH fl 2)4 NH
8 6 n 0 0 8 0 1 n
m P
R5 R7Y __________ X ______ R5 O
01/
R3 R3
I I H
---(CH2)4"--C-NH-C R1 C ___________________________________________ NH C C 0
R4 0 C CH N-C R1-C
H 0 0 H n H " ii 0 II II
P 0 0 0 n
Formula (XII)
wherein, X is selected from the group consisting of (CI-C18) alkylene,
substituted alkylene, (C3-
C8) cycloalkylene, substituted cycloalkylene, 5-6 membered heterocyclic system
containing 1-3
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heteroatoms selected from the group 0, N, and S, substituted heterocyclic, (C2-
C18) alkenyl,
substituted alkenyl, alkynyl, substituted alkynyl, (C6 - Cm) aryl, substituted
aryl, heteroaryl,
substituted heteroaryl, allcylaryl, substituted allcylaryl, arylalkynyl,
substituted arylalkynyl,
arylalkenyl, substituted arylalkenyl, arylalkynyl, substituted arylalkynyl,
wherein the
substituents are selected from the group consisting of H, F, Cl, Br, I, (Ci-
C6) alkyl, -CN,
-NO2,, ¨OH, -0(C1-C6) alkyl, -S(Ci-C6) alkyl, -S[(=0)(CI-C6) alkyl], -
S[(02)(Ci-C6) alkyl],
-CR=0)(Ci-C6) alkyl], CF3,-0[(C0)-(CI-C6) alkyl], -S(02)[N(R9R19)], -
NH[(C=0)(C1-C6)
alkyl], -NH(C=0)N(R9R1 ), wherein R9 and R19 are independently H or (C1-C6)
alkyl, and -
N(R11R12), wherein R11 and R12 are independently selected from (C2-C20)
alkylene and (C2-C2o)
alkenylene.
100511 In yet another embodiment, the intraocular polymer delivery
composition contains
four molecules of the polymer, except that only two of the four molecules omit
112 and are
crosslinked to provide a single ¨R5-X-R5- conjugate.
[0052] The term "aryl" is used with reference to structural formulae herein
to denote a phenyl
radical or an ortho-fused bicyclic carbocyclic radical having about nine to
ten ring atoms in
which at least one ring is aromatic. In certain embodiments, one or more of
the ring atoms can
be substituted with one or more of nitro, cyano, halo, trifluoromethyl, or
trifluoromethoxy.
Examples of aryl include, but are not limited to, phenyl, naphthyl, and
nitrophenyl.
100531 The term "alkenylene" is used with reference to structural formulae
herein to mean a
divalent branched or unbranched hydrocarbon chain containing at least one
unsaturated bond in
the main chain or in a side chain.
[00541 The molecular weights and polydispersities herein are determined by
gel permeation
chromatography (GPC) using polystyrene standards. More particularly, number
and weight
average molecular weights (Mn and MO are determined, for example, using a
Model 510 gel
permeation chromatography (Water Associates, Inc., Milford, MA) equipped with
a high-
pressure liquid chromatographic pump, a Waters 486 UV detector and a Waters
2410 differential
refractive index detector. Tetrahydrofuran (THF), N,N-dimethylformamide (DMF)
or N,N-
dimethylacetamide (DMAc) is used as the eluent (1.0 mL/min). Polystyrene or
poly(methyl
methacrylate) standards having narrow molecular weight distribution were used
for calibration.
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100551 Methods for making polymers of structural formulas containing a
a¨amino acid in the
general formula are well known in the art. For example, for the embodiment of
the polymer of
structural formula (I) wherein R4 is incorporated into an a¨amino acid, for
polymer synthesis the
a¨amino acid with pendant R3 canbe converted through esterification into a bis-
a,0-diamine,
for example, by condensing the a¨amino acid containing pendant R3 with a diol
HO¨R4-0H.
As a result, di-ester monomers with reactive a,0-amino groups are formed.
Then, the bis-a,0-
diamine is entered into a polycondensation reaction with a di-acid such as
sebacic acid, or bis-
activated esters, or bis-acyl chlorides, to obtain the final polymer having
both ester and amide
bonds (PEA). Alternatively, for example, for polymers of structure (I),
instead of the di-acid, an
activated di-acid derivative, e.g., bis-para-nitrophenyl diester, can be used
as an activated di-
acid. Additionally, a bis-di-carbonate, such as bis(p-nitrophenyl)
dicarbonate, can be used as the
activated species to obtain polymers containing a residue of a di-acid. In the
case of PEUR
polymers, a final polymer is obtained having both ester and urethane bonds.
[0056j More particularly, synthesis of the unsaturated poly(ester-amide)s
(UPEAs) useful as
biodegradable polymers of the structural formula (I) as disclosed above will
be described,
wherein
0
0 0
(a) iscõ18.-ir
0
and/or (b) R4 is ¨CH2-CH=CH-CH2- . In cases where (a) is present and (b) is
not present, R4 in
(I) is ¨C4F18- or ¨C6H12-. In cases where (a) is not present and (b) is
present, RI in (I) is ¨C4I-18-
or ¨C8H
100571 The UPEAs can be prepared by solution polycondensation of either (I)
di-p-toluene
sulfonic acid salt of bis(a-amino acid) di-ester of unsaturated diol and di-p-
nitrophenyl ester of
saturated dicarboxylic acid or (2) di-p-toluene sulfonic acid salt of bis (a-
amino acid) diester of
saturated diol and di-nitrophenyl ester of unsaturated dicarboxylic acid or
(3) di-p-toluene
sulfonic acid salt of bis(a-amino acid) diester of unsaturated diol and di-
nitrophenyl ester of
unsaturated dicarboxylic acid.
100581 The aryl sulfonic acid salts of diamines are known for use in
synthesizing polymers
containing amino acid residues. The p-toluene sulfonic acid salts are used
instead of the free
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22
diamines because the aryl sulfonic salts of bis (a-amino acid) diesters are
easily purified through
recrystallization and render the amino groups as less reactive ammonium
tosylates throughout
workup. In the polycondensation reaction, the nucleophilic amino group is
readily revealed
through the addition of an organic base, such as triethylamine, reacts with
bis-etectrophilic
monomer, so the polymer product is obtained in high yield.
L00591 Bis-electrophilic monomers, for example, the di-p-nitrophenyl esters
of unsaturated
dicarboxylic acid, can be synthesized from p-nitrophenyl and unsaturated
dicarboxylic acid
chloride, e.g., by dissolving triethylamine and p-nitrophenol in acetone and
adding unsaturated
dicarboxylic acid chloride dropwise with stirring at -78 C and pouring into
water to precipitate
product. Suitable acid chlorides included fumaric, maleic, mesaconic,
citraconic, glutaconic,
itaconic, ethenyl-butane dioic and 2-propenyl-butanedioic acid chlorides. For
polymers of
structure (V) and (VI), bis-p-nitrophenyl dicarbonates of saturated or
unsaturated diols are used
as the activated monomer. Dicarbonate monomers of general structure (XIII) are
employed for
polymers of structural formula (V) and (VI),
00
II JI
Formula (XIII)
wherein each R5 is independently (C6 -C10) aryl optionally substituted with
one or more nitro,
cyano, halo, trifluoromethyl, or trifluoromethoxy; and R6 is independently (C2
-C2o) allcylene or
(C2 ¨C20) alkyloxy, or (C2 -C20) alkenylene.
100601 Suitable therapeutic diol compounds that can be used to prepare
bis(a-amino acid)
diesters of therapeutic diol monomers, or bis(carbonate) of therapeutic di-
acid monomers, for
introduction into the invention intraocular polymer delivery compositions
include naturally
occurring therapeutic diols, such as 17-13-estradiol, a natural and endogenous
hormone, useful in
preventing restenosis and tumor growth. The procedure for incorporation of a
therapeutic diol,
such as an ophthalmologic diol as disclosed herein, into the backbone of a
PEA, PEUR or PEU
polymer is illustrated in this application by Example 8, in which active
steroid hormone 1713-
estradiol containing mixed hydroxyls - secondary and phenolic - is introduced
into the backbone
of a PEA polymer. When the PEA polymer is used to fabricate particles or
solids and the
particles or solids are implanted intraocularly, the therapeutic diol is
released from the particles
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23
or solids at a controlled rate. In the present invention, the preferred
therapeutic diol for
incorporation into the backbone of the polymer is an ophthalmologic diol.
100611 Due to the versatility of the PEA, PEUR and PEU polymers used in the
invention
compositions, the amount of the therapeutic diol or di-acid incorporated in a
polymer backbone
can be controlled by varying the proportions of the building blocks of the
polymer. For
example, depending on the composition of the PEA, loading of up to 40% w/w of
1713-estradiol
can be achieved. Two different regular, linear PEAs with various loading
ratios of 1713-estradiol
illustrate this concept in Scheme 1 below:
-
0 o 9 o
--4-NH-c H-a-0 .0,õ,,, MP 0-6-CH-NH-c-(cH04-ah
9H2 CH2
' CH(CH3)2 CH(CH3)2
Polyesteramide of formula ( I ), based on 17,beta-estradiole, L-leucine and
adipic acid
with 40 % w/w estradiol on polymer
9 0 0 0-
NHIH2 H-C-0
1
CH(CH3)2
..
..
1,=== o-c-cH2-NH-C-H2
-6-
CH(CH3)2 -3n/4
-[CC-(CH2)4-2
-HN-HCICH2)4¨NH
I
CO0C2H5 n/4
Polyesteramide of formula ( IV ), based on I 7,beta-estradiole, L-Ieucine, L-
lysine ethyl ester
and adipic acid with 38 % w/w estradiol load
Scheme 1 .
Similarly, the loading of the therapeutic diol into PEUR and PEU polymer can
be varied by
varying the amount of two or more building blocks of the polymer.
100621 Suitable ophthalmologic synthetic steroid based diols, based on
testosterone or
cholesterol, that can be dispersed in or incorporated into the backbones of
the polymers used in
the invention intraocular implant compositions include such compounds a
hydrocortisone,
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24
fluorocortisone, cortisone, aldosterone, betamethasone, prednisolone,
dexamethasone,
fluocinolone acetonide, and the like.
100631 Additional ophthalmologic diol compounds that can be used to prepare
an amide
linkage in the PEA polymer compositions of the invention include, for example,
latanoprost,
brimatoprost, travoprost, cidofovir, pencyclovir, and the like.
100641 Additional, synthetic steroid based therapeutic diols based on
testosterone or
cholesterol, such as 4-androstene-3, 17 diol (4-androstenediol), 5-androstene-
3, 17 diol (5-
androstenediol), 19-nor5-androstene-3, 17 diol (19-norandrostenediol) are also
suitable for
incorporation into the backbone of PEA. PEUR and PEU polymers according to
this invention.
For example, therapeutic diol compounds suitable for use in preparation of the
invention
intraocular polymer particle or solid delivery compositions include, for
example, amikacin;
amphotericin B; apicycline; apramycin; arbekacin; azidamfenicol;
bambermycin(s); butirosin;
carbomycin; cefpiramide; chloramphenicol; chlortetracycline; clindamycin;
clomocycline;
demeclocycline; diathymosulfone; dibekacin, dihydrostreptomycin;
dirithromycin; doxycycline;
erythromycin; fortimicin(s); gentamycin(s); glucosulfone solasulfone;
guamecycline;
isepamicin; josamycin; kanamycin(s); leucomycin(s); lincomycin; lucensomycin;
lymecycline;
meclocycline; methacycline; micronomycin; midecamycin(s); minocycline;
mupirocin;
natamycin; neomycin; netilmicin; oleandomycin; oxytetracycline; paromycin;
pipacycline;
podophyllinic acid 2-ethylhydrazine; primycin; ribostamycin; rifamide;
rifampin; rafamycin SV;
rifapentine; rifaxi min; ristocetin; rokitamycin; rolitetracycline;
rasaramycin; roxithromycin;
sancycline; sisomicin; spectinomycin; spiramycin; streptomycin; teicoplanin;
tetracycline;
thiamphenicol; theiostrepton; tobramycin; trospectomycin; tuberactinamycin;
vancomycin;
candicidin(s); chlorphenesin; dermostatin(s); filipin; fungichromin;
kanamycin(s);
leucomycins(s); lincomycin; lvcensomycin; lymecycline; meclocycline;
methacycline;
micronomycin; midecamycin(s); minocycline; mupirocin; natamycin; neomycin;
netilmicin;
oleandomycin; oxytetracycline; paramomycin; pipacycline; podophyllinic acid 2-
ethylhydrazine;
priycin; ribostamydin; rifamide; rifampin; rifamycin SV; rifapentine;
rifaximin; ristocetin;
rokitamycin; rolitetracycline; rosaramycin; roxithromycin; sancycline;
sisomicin; spectinomycin;
spiramycin; strepton; otbramycin; trospectomycin; tuberactinomycin;
vancomycin;
candicidin(s); chlorphenesin; dermostatin(s); filipin; fungichromin;
meparticin; mystatin;
oligomycin(s); erimycinA; tubercidin; 6-azauridine; aclacinomycin(s);
ancitabine; anthramycin;
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azacitadine; bleomycin(s) carubicin; carzinophillin A; chlorozotocin;
chromomcin(s);
doxifluridine; enocitabine; epirubicin; gemcitabine; mannomustine; menogaril;
atorvasi
pravastatin; clarithromycin; leuproline; paclitaxel; mitobronitol; mitolactol;
mopidamol;
nogalamycin; olivomycin(s); peplornycin; pirarubicin; prednimustine;
puromycin; ranimustine;
tubercidin; vinesine; zorubicin; coumetarol; dicoumarol; ethyl biscoumacetate;
ethylidine
dicoumarol; iloprost; taprostene; tioclomarol; amiprilose; romurtide;
sirolimus (rapamycin);
tacrolimus; salicyl alcohol; bromosaligenin; ditazol; fepradinol; gentisic
acid; glucamethacin;
olsalazine; S-adenosylmethionine; azithromycin; salmeterol; budesonide;
albuteal; indinavir;
fluvastatin; streptozocin; doxorubicin; daunorubicin; plicamycin; idarubicin;
pentostatin;
metoxantrone; cytarabine; fludarabine phosphate; floxuridine; cladriine;
capecitabien; docetaxel;
etoposide; topotecan; vinblastine; teniposide, and the like. The therapeutic
diol can be selected
to be either a saturated or an unsaturated diol.
100651 Suitable naturally occurring and synthetic therapeutic di-acids that
can be used to
prepare an amide linkage in the PEA polymer compositions of the invention
include, for
example, bambermycin(s); benazepril; carbenicillin; carzinophillin A;
cefixime; cefininox
cefpimizole; cefodizime; cefonicid; ceforanide; cefotetan; ceftazidime;
ceftibuten; cephalosporin
C; cilastatin; denopterin; edatrexate; enalapril; lisinopril; methotrexate;
moxalactam; nifedipine;
olsalazine; penicillin N; ramipril; quinacillin; quinapril; temocillin;
ticarcillin; Tomudexe (N-
[[5-[[(1,4-Dihydro-2-methyl-4-oxo-6-quinazolinyl)methyl] methylamino]-2-
thienylicarbonyll-L-
glutamic acid), and the like. The safety profile of naturally occurring
therapeutic di-acids is
believed to surpass that of synthetic therapeutic di-acids. The therapeutic di-
acid can be either a
saturated or an unsaturated di-acid.
100661 The chemical and therapeutic properties of the above described
ophthalmologic and
other therapeutic diols and di-acids as inhibitors of macular degeneration,
tumor inhibitors,
cytotoxic antimetabolites, antibiotics, and the like, are well known in the
art and detailed
descriptions thereof can be found, for example, in thel 3th Edition of The
Merck Index
(Whitehouse Station, N.J., USA).
100671 The di-aryl sulfonic acid salts of diesters of cc-amino acid and
unsaturated diol can be
prepared by admixing a-amino acid, e.g., p-aryl sulfonic acid monohydrate and
saturated or
unsaturated diol in toluene, heating to reflux temperature, until water
evolution is minimal, then
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26
cooling. The unsaturated diols include, for example, 2-butene-1,3-diol and
1,18-octadec-9-en-
diol.
100681 Saturated di-p-nitrophenyl esters of dicarboxylic acid and saturated
di-p-toluene
sulfonic acid salts of bis-a -amino acid esters can be prepared as described
in U.S. Patent No.
6,503,538 Bl.
100691 Synthesis of the unsaturated poly(ester-amide)s (UPEAs) useful as
biodegradable
polymers of the structural formula (I) as disclosed above will now be
described. UPEAs having
the structural formula (I) can be made in similar fashion to the compound
(VII) of U. S. Patent
No. 6,503,538 B1, except that R4 of (III) of 6,503,538 and/or RI of (V) of
6,503,538 is (C2-C20)
alkenylene as described above. The reaction is carried out, for example, by
adding dry
triethylamine to a mixture of said (III) and (IV) of 6,503,538 and said (V) of
6,503,538 in dry
N,N-dimethylacetamide, at room temperature, then increasing the temperature to
80 C and
stirring for 16 hours, then cooling the reaction solution to room temperature,
diluting with
ethanol, pouring into water, separating polymer, washing separated polymer
with water, drying
to about 30 C under reduced pressure and then purifying up to negative test on
p-nitrophenol
and p-toluene sulfonate. A preferred reactant (IV) of 6,503,538 is p-toluene
sulfonic acid salt of
Lysine benzyl ester, the benzyl ester protecting group is preferably removed
from (II) to confer
biodegradability, but it should not be removed by hydrogenolysis as in Example
22 of U.S.
Patent No. 6,503,538 because hydrogenolysis would saturate the desired double
bonds; rather
the benzyl ester group should be converted to an acid group by a method that
would preserve
unsaturation. Alternatively, the lysine reactant (IV) of 6,503,538 can be
protected by a
protecting group different from benzyl that can be readily removed in the
finished product while
preserving unsaturation, e.g., the lysine reactant can be protected with t-
butyl (i.e., the reactant
can be t-butyl ester of lysine) and the t-butyl can be converted to H while
preserving
unsaturation by treatment of the product (II) with acid.
100701 A working example of the compound having structural formula (I) is
provided by
substituting p-toluene sulfonic acid salt of bis(L-phenylalanine) 2-butene-1,4-
diester for (III) in
Example 1 of 6,503,538 or by substituting di-p-nitrophenyl fumarate for (V) in
Example I of
6,503,538 or by substituting the p-toluene sulfonic acid salt of bis(L-
phenylalanine) 2-butene-
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27
1,4-diester for III in Example 1 of 6,503,538 and also substituting bis-p-
nitrophenyl fumarate for
(V) in Example 1 of 6,503,538.
[00711 In unsaturated compounds having either structural formula (I) or
(IV), the following
hold. An amino substituted aminoxyl (N-oxide) radical bearing group, e.g., 4-
amino TEMPO,
can be attached using carbonyldiimidazol, or suitable carbodiimide, as a
condensing agent.
Bioactive agents, as described herein, can be attached via the double bond
functionality.
Hydrophilicity can be imparted by bonding to poly(ethylene glycol) diacrylate.
100721 In yet another aspect, the PEA and PEUR polymers contemplated for
use in forming
the invention polymer particle delivery systems include those set forth in
U.S. Patent Nos.
5,516, 881; 6,476,204; 6,503,538; and in U.S. Application Nos. 10/096,435;
10/101,408;
10/143,572; and 10/194,965; the entire contents of each of which is
incorporated herein by
reference.
[00731 The biodegradable PEA, PEUR and PEU polymers can contain from one to
multiple
different ophthalmologic compounds and cc-amino acids per polymer molecule and
preferably
have weight average molecular weights ranging from 10,000 to 125,000; these
polymers and
copolymers typically have intrinsic viscosities at 25 C, as determined by
standard viscosimetric
methods, ranging from 0.3 to 4.0, for example, ranging from 0.5 to 3.5.
[00741 PEA and PEUR polymers contemplated for use in the practice of the
invention can be
synthesized by a variety of methods well known in the art. For example,
tributyltin (IV)
catalysts are commonly used to form polyesters such as poly(c-caprolactone),
poly(glycolide),
poly(lactide), and the like. However, it is understood that a wide variety of
catalysts can be used
to form polymers suitable for use in the practice of the invention.
100751 Such poly(caprolactones) contemplated for use have an exemplary
structural formula
(XIV) as follows:
0-8--(0H2)51¨
Formula (XIV)
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[0076] Poly(glycolides) contemplated for use have an exemplary structural
formula (XV) as
follows:
OH
c C _________________________________________
'In
Formula (XV)
[0077] Poly(lactides) contemplated for use have an exemplary structural
formula (XVI) as
follows:
0 Melo
Formula (XVI)
[0078] An exemplary synthesis of a suitable poly(lactide-co-c-caprolactone)
including an
aminoxyl moiety is set forth as follows. The first step involves the
copolymerization of lactide
and c-caprolactone in the presence of benzyl alcohol using stannous octoate as
the catalyst to
form a polymer of structural formula (XVII).
0 0
=
yjLo
cH,cni +Me n 0 m
y ¨Me
0
=
CH2 0-tg CI {¨ 25 ¨g (CH ) OFH
-
Me n
Formula (XVII)
[0079] The hydroxy terminated polymer chains can then be capped with maleic
anhydride to
form polymer chains having structural formula (XVIII):
= CH OPCs 01 [2 (CH ) 01-g
2 C C OH
--
Me n m H H
Formula (XVIII)
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100801 At this point, 4-amino-2,2,6,6-tetramethylpiperidine-1-oxy can be
reacted with the
carboxylic end group to covalently attach the aminoxyl moiety to the copolymer
via the amide
bond which results from the reaction between the 4-amino group and the
carboxylic acid end
group. Alternatively, the maleic acid capped copolymer can be grafted with
polyacrylic acid to
provide additional carboxylic acid moieties for subsequent attachment of
further aminoxyl
groups.
100811 In unsaturated compounds having structural formula (VII) for PEU,
the following
hold: An amino substituted aminoxyl (N-oxide) radical bearing group e.g., 4-
amino TEMPO,
can be attached using carbonyldiimidazole, or suitable carbodiimide, as a
condensing agent.
) Additional bioactive agents, and the like, as described herein, optionally
can be attached via the
double bond functionality provided that the therapeutic diol residue in the
polymer composition
does not contain a double or triple bond.
[00821 For example, the invention high molecular weight semi-crystalline
PEUs having
structural formula (VII) can be prepared inter-facially by using phosgene as a
bis-electrophilic
monomer in a chloroform/water system, as shown in the reaction scheme (2)
below:
1. Na2co3 / H20
H 0 9 H 2. CICOCI / CHC13
HOTos.H2N¨C-8-0-R1-0-C-C-NH2.Tos0H ( VII )
R3 R3
Scheme 2
Synthesis of copoly(ester ureas) (PEUs) containing L-Lysine esters and having
structural
formula (VII) can be carried out by a similar scheme (3):
HO 9 H11
m HOTos.H2N¨C¨C-O-R1-0-C-C-NH2.Tos0H + p HOTos.H2N-C-(CH2)4¨NH2.Tos0H
123 R3
C-O-R2
0
1. Na2CO3 / H20
2. CICOCI / CHC13
__________________ = ( VIII )
Scheme 3
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A 20% solution of phosgene (CICOCI) (highly toxic) in toluene, for example
(commercially
available (Fluka Chemie, GMBH, Buchs, Switzerland), can be substituted either
by diphosgene
(trichloromethylchloroformate) or triphosgene (bis(trichloromethyl)carbonate).
Less toxic
carbonyldiimidazole can be also used as a bis-electrophilic monomer instead of
phosgene, di-
phosgene, or tri-phosgene.
[0083] General Procedure for Synthesis of PEUs It is necessary to use
cooled solutions of
monomers to obtain PEUs of high molecular weight. For example, to a suspension
of di-p-
toluenesulfonic acid salt of bis(a-amino acid)-am-alkylene diester in 150 mL
of water,
anhydrous sodium carbonate is added, stirred at room temperature for about 30
minutes and
cooled to about 2 - 0 C, forming a first solution. In parallel, a second
solution of phosgene in
chloroform is cooled to about 15 -10 C. The first solution is placed into a
reactor for interfacial
polycondensation and the second solution is quickly added at once and stirred
briskly for about
15 min. Then chloroform layer can be separated, dried over anhydrous Na2SO4,
and filtered.
The obtained solution can be stored for further use.
100841 All the exemplary PEU polymers fabricated were obtained as solutions
in chloroform
and these solutions are stable during storage. However, some polymers, for
example, 1-Phe-4,
become insoluble in chloroform after separation. To overcome this problem,
polymers can be
separated from chloroform solution by casting onto a smooth hydrophobic
surface and allowing
chloroform to evaporate to dryness. No further purification of obtained PEUs
is needed. The
yield and characteristics of exemplary PEUs obtained by this procedure are
summarized in Table
I herein.
[0085] General Procedure for Preparation of porous PEUs. Methods for making
the PEU
polymers containing a-amino acids in the general formula will now be
described. For example,
for the embodiment of the polymer of formula (1) or (III), the a-amino acid
can be converted into
a bis(a-amino acid)-am-diol-diester monomer, for example, by condensing the a-
amino acid
with a diol HO-111-0H. As a result, ester bonds are formed. Then, acid
chloride of carbonic
acid (phosgene, diphosgene, triphosgene) is entered into a polycondensation
reaction with a di-p-
toluenesulfonic acid salt of a bis(a-amino acid) -alkylene diester to obtain
the final polymer
having both ester and urea bonds. In the present invention, at least one
therapeutic diol can be
used in the polycondensation protocol.
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[0086] The unsaturated PEUs can be prepared by interfacial solution
condensation of di-p-
toluenesulfonate salts of bis(a-amino acid)-alkylene diesters, comprising at
least one double
bond in RI. Unsaturated diols useful for this purpose include, for example, 2-
butene-1,4-diol and
1,18-octadec-9-en-diol. Unsaturated monomer can be dissolved prior to the
reaction in alkaline
water solution, e.g. sodium hydroxide solution. The water solution can then be
agitated
intensely, under external cooling, with an organic solvent layer, for example
chloroform, which
contains an equimolar amount of monomeric, dimeric or trimeric phosgene. An
exothermic
reaction proceeds rapidly, and yields a polymer that (in most cases) remains
dissolved in the
organic solvent. The organic layer can be washed several times with water,
dried with
anhydrous sodium sulfate, filtered, and evaporated. Unsaturated PEUs with a
yield of about
75%-85% can be dried in vacuum, for example at about 45 C.
[0087] To obtain a porous, strong material, L-Leu based PEUs, such as 1-L-
Leu-4 and 1-L-
Leu-6, both of formula (VII), can be fabricated using the general procedure
described below.
Such procedure is less successful in formation of a porous, strong material
when applied to L-
Phe based PEUs.
[0088] The reaction solution or emulsion (about 100 mL) of PEU in
chloroform, as obtained
just after interfacial polycondensation, is added dropwise with stirring to
1,000 mL of about 80 -
85 C water in a glass beaker, preferably a beaker made hydrophobic with
dimethyldichlorsilane
to reduce the adhesion of PEU to the beaker's walls. The polymer solution is
broken in water
into small drops and chloroform evaporates rather vigorously. Gradually, as
chloroform is
evaporated, small drops combine into a compact tar-like mass that is
transformed into a sticky
rubbery product. This rubbery product is removed from the beaker and put into
hydrophobized
cylindrical glass-test-tube, which is thermostatically controlled at about 80
C for about 24
hours. Then the test-tube is removed from the thermostat, cooled to room
temperature, and
broken to obtain the polymer. The obtained porous bar is placed into a vacuum
drier and dried
under reduced pressure at about 80 C for about 24 hours. In addition, any
procedure known in
the art for obtaining porous polymeric materials can also be used.
[0089] Properties of high-molecular-weight porous PEUs made by the above
procedure
yielded results as summarized in Table 2.
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Table 1 Properties of PEU Polymers of Formula (VII) and (VIII)
PEU* Yield gred a) Afw b) Ain b) man, b) Tg Tm
(A) IdL/g] I C1 1 C]
1-L-Leu-4 80 0.49 84000 45000 1.90 67 103
1-L-Leu-6 82 0.59 96700 50000 1.90 64 126
1-L-Phe-6 77 0.43 60400 34500 1.75
167
[1-L-Leu-6]0 75- [1-L- 84 0.31 64400 43000 1.47 34
114
Lys(0Bn)]o 25
1-L-Leu-DAS 57 0.28 55700d) 27700d) 2.1d) 56
165
*PEUs of general formula (VII), where,
1-L-Leu-4: R4 = (CH2)4, R3 = i-C4H9
1-L-Leu-6: R4 = (CH2)6, R3 = i-C4H9
1-L-Phe-6:.R4 = (CH2)6, R3 = -CH2-C6H5.
1-L-Leu-DAS: R4 = 1,4:3,6-dianhydrosorbitol, R3 = i-C4H
a) Reduced viscosities were measured in DMF at 25 C and a concentration 0.5
g/dL
b) GPC Measurements were carried out in DMF, (PMMA)
c) Tg taken from second heating curve from DSC Measurements (heating rate10
C/min).
GPC Measurements were carried out in DMAc, (PS)
[0090] Tensile strength of illustrative synthesized PEUs was measured and
results are
summarized in Table 2. Tensile strength measurement was obtained using
dumbbell-shaped
PEU films (4 x 1.6 cm), which were cast from chloroform solution with average
thickness of
0.125 mm and subjected to tensile testing on tensile strength machine
(Chatillon TDC200)
integrated with a PC using Nexygen FM software (Amtek, Largo, FL) at a
crosshead speed of 60
mm/min. Examples illustrated herein can be expected to have the following
mechanical
properties:
. A glass transition temperature in the range from about 30 C to about 90 C,
for
example, in the range from about 35 C to about 70 C;
2. A film of the polymer with average thickness of about 1.6 cm will have
tensile
stress at yield of about 20 Mpa to about 150 Mpa, for example, about 25 Mpa to
about 60 Mpa;
3. A film of the polymer with average thickness of about 1.6 cm will have a
percent
elongation of about 10 % to about 200%, for example about 50 % to about 150%;
and
4. A film of the polymer with average thickness of about 1.6 cm will have a
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33
Young's modulus in the range from about 500 MPa to about 2000 MPa. Table 2
below
summarizes the properties of exemplary PEUs of this type.
Table 2 Mechanical Properties of PEUs
Tga) Tensile Stress Young's
Percent
at Yield Polymer designationeld Modulus
( C) Elongation (%)
(MPa) (MPa)
1-L-Leu-6 64 21 114 622
[1- L-Leu-610 75- [1-L-Lys(0Bn)]o 25 34 25 159 915
100911 In another embodiment, the invention provides solid polymer
intraocular delivery
compositions comprising one or more solid layers comprising at least one
biodegradable,
biocompatible polymer as a carrier layer into which is dispersed, mixed,
dissolved,
homogenized, or matrixed (i.e., "dispersed") at least one ophthalmologic
agent. Two or more
ophthalmologic agents may also be dispersed into a carrier layer, or invention
compositions
having more than one carrier layer may have two or more ophthalmologic agents
dispersed into
separate carrier layers therein.
100921 In one embodiment, the invention provides a solid polymer
intraocular delivery
composition for implant intraocularly, said composition comprising at least
one biodegradable,
biocompatible polymer having a chemical formula described by general
structural formulas (I) -
(IV-VIII) as described herein into which is dispersed at least one
ophthalmologic agent for
release at a controlled rate over a considerable period of time, for example,
over a period of three
months to about twelve months. Optionally, an additional bioactive agent, as
described herein,
may also be dispersed in the at least one polymer. The ophthalmologic agent
and any optional
bioactive agent present therein is released from the composition in situ
(i.e., intraocularly) as a
result of biodegradation of the various polymer layers in the composition.
100931 The solid polymer intraocular delivery composition may further
comprise at least one
coating layer of a biodegradable, biocompatible polymer, which may or may not
have dispersed
therein such an ophthalmologic agent. The purpose of the coating layer of
polymer, for example
a pure polymer shell, is to slow release of the ophthalmologic agent contained
in the
composition. The PEA, PEUR and PEU polymers of formulas (I) and (IV-VIII)
described
herein readily absorb water, allowing hydrophilic molecules to readily diffuse
therethrough.
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This characteristic makes such PEA, PEUR and PEU polymers suitable for use as
a coating on
the invention solid polymer compositions to control release rate of the at
least one bioactive
agent therefrom.
[0094] Rate of release of the at least one ophthalmologic agent from the
invention solid
polymer intraocular delivery compositions can be controlled, not only by
selection of the
polymers in various layers of the composition, but by adjusting the coating
thickness and
density, as well as by the number of coating layers contained in the invention
composition.
Density of the coating layer can be adjusted by adjusting loading of the
active agent(s) in the
coating layer. For example, when the coating layer contains no bioactive
agent, the polymer
coating layer is densest, and an ophthalmologicall, or optional bioactive,
agent from the interior
of the composition elutes through the coating layer most slowly. By contrast,
when an
ophthalmological!, or optional bioactive, agent is dispersed within (i.e. is
mixed or "matrixed"
with) biodegradable, biocompatible polymer in the coating layer, the coating
layer becomes
porous once any active agent in the coating layer has eluted out, starting
from the outer surface
of the coating layer. A porous coating layer once formed by this process, an
ophthalmologic
agent in the carrier layer(s) of the solid composition can elute at an
increased rate. The higher
the active agent loading in the coating layer, the lower the density of the
coating layer and the
higher the elution rate. Although loading of active agent in the coating layer
can be lower or
higher than that in the carrier layer(s), for slowest sustained delivery of an
ophthalmologic agent
at a controlled rate, the coating layer is a pure polymer shell. There may be
multiple coating
layers as well as multiple carrier layers in the invention composition.
100951 Accordingly, in one embodiment the invention provides a solid
polymer composition
for controlled release of an ophthalmologic agent, said composition comprising
a carrier layer
containing at least one ophthalmologic agent dispersed in a biodegradable,
biocompatible
polymer having a structural formula described by structural formulas (I) and
(IV-VIII), and at
least one coating layer that covers the carrier layer, wherein the coating
layer comprises a
biodegradable, biocompatible polymer, such as those described by structural
formulas (I) and
(IV). For convenience in manufacture, the polymer of the coating layer may be
the same as the
polymer of the carrier layer.
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100961 However, it has been discovered that, during manufacture, solvent in
the polymer
dispersion used to make the coating layer(s) of the invention solid polymer
intraocular delivery
composition tends to elute a matrixed ophthalmologic agent out of the carrier
layer, even though=
the carrier layer has been dried prior to application of the coating layer. To
prevent the solvent
used in applying the coating layer from robbing the carrier layer of its load
of bioactive agent,
the invention composition may further comprise a barrier layer between a
carrier layer and each
of one or more coating layers. The barrier layer is made using a liquid
polymer that will not
dissolve in the solvent used in the polymer solution or dispersion that lays
down the coating
layer in the invention composition, but which barrier layer polymer dissolves
in physiologic
conditions, for example in the presence of aqueous conditions and physiologic
enzymes. Thus,
the barrier layer(s), as well as the coating layer(s) of the invention
composition, aid in
controlling the release rate of the ophthalmologic agent from the carrier
polymer layer.
100971 In certain embodiments, invention solid polymer intraocular delivery
compositions
can have one or multiple sets of the barrier layer and coating layer, with the
coating layer being
exterior in the final composition to each successive set. Consequently, the
carrier layer is
sequestered within the protective sets of barrier layer and coating layer. For
example, one to ten
sets of the barrier layer and coating layer may be present, e.g., one to eight
sets, with the number
of sets determining the rate of release of the ophthalmologic agent in the
carrier layer from the
composition. A larger number of sets provide for a slower release rate and a
lesser number of
sets provides for a faster release rate.
100981 In one embodiment, the sets of barrier layer and coating layer may
be aligned in a
parallel configuration on either side of the carrier layer in a sandwich
structure, with the carrier
layer at the center of two sets of barrier layer and coating layer on either
side. In another
embodiment, succeSsive outwardly lying layers of the multiple sets of layers
encompass all
interior layers to form an onionskin structure with the carrier layer
sequestered at the center of
the composition. Thickness of the carrier layer and concentration of the one
or more
ophthalmologic agents (and optional bioactive agents) in the carrier layer
determine total dosage
of each agent that the composition can deliver when implanted. After implant
or in vitro
exposure to physiological conditions (e.g. water and enzymes) the outward most
layers of
coating and barrier layer begin to biodegrade as encountered and the matrixed
bioactive agent(s)
elutes from the sequestered carrier layer. As the concentration of bioactive
agent in the interior
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carrier layer diminishes, the surface area of the composition will tend to
diminish as well due to
biodegradation, with both effects tending to slow the rate of release of the
bioactive agent in a
controlled manner.
100991 In another embodiment, the invention composition comprises multiple
sets of carrier
and barrier layers arranged in an onionskin structure with a single exterior
coating of pure
biodegradable, biocompatible polymer or alternating layers of barrier layer
and coating as
described above. In this embodiment, water and enzymes from the environment,
e.g.,
intraocular tissue surroundings, successively dissolve the outward most
coating(s) and barrier
layer(s) encountered to elute ophthalmologic agent from successive carrier
layers, thereby
releasing the ophthalmologic agent(s), which can either be matrixed in the
polymer or
incorporated into the polymer backbone. If the concentration of ophthalmologic
active agent is
substantially constant in the multiple layers of the onionskin structure, the
rate of delivery will
diminish in proportion as the surface area of the onionskin structure
diminishes. To accomplish
a more constant rate of delivery of the matrixed bioactive agent(s), it is
recommended in this
embodiment to utilize a gradient of concentration of bioactive agent in the
carrier layers in the
onionskin structure, with a higher concentration being used in inner carrier
layers in proportion
to the diminishing surface area of the composition. Those skilled in the art
will understand that
routine principles of fluid dynamics can be used to calculate the theoretical
rate of delivery and
to obtain a substantially constant rate of delivery as the onionskin structure
diminishes in surface
area, if desired.
101001 In yet another embodiment, the invention solid polymer intraocular
delivery
composition comprises a carrier layer with additional polymer layers arranged
in an onionskin or
sandwich structure about the carrier layer. In one alternative, there can be a
single exterior
coating layer of pure biodegradable, biocompatible polymer formed by spraying
the carrier layer
and coating layer, for example, as an aerosol. This structure favored for
compositions having a
thickness of 0.1 mm to 2.5 mm in thickness for rapid release of the
ophthalmologic agent(s). In
another alternative, several coating layers extend outwardly from the core
carrier layer in an
onionskin or sandwich structure. Alternatively still, to achieve a more
constant rate of delivery,
multiple carrier layers can be employed, provided that each carrier layer is
subsequently coated
with a coating layer. To effect a gradient of concentration of bioactive
molecule(s) in the carrier
layer(s) in the onionskin structure, a higher concentration of ophthalmologic
molecule(s) is used
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in inner carrier layers in proportion to the outer carrier layers, such that
the rate of release of
ophthalmologic molecule(s) can be maintained as the surface area of the
composition diminishes
during the biodegradation of the invention composition.
101011 The invention solid intraocular polymer delivery composition
typically has a three-
dimensional shape compatible with the properties of the polymer and which
meets the
therapeutic and delivery requirements for a given ophthalmologic agent and
route of
administration., such as a wafer, sheet or film, ball, disc, cylinder, fiber,
tube, and the like.
Constructs will be sized according to delivery requirements and location of
administration. For
example, for subconjunctival administration, the size of the construct will be
preferably no larger
than about a I mm by 1 mm by 7 mm rectangle or cylinder suitable for injection
through a
hypodermic needle. The bore requirements of the hypodermic needle should
coincide with the
route and location of administration, e.g., for subconjunctival administration
the needle bore
should be about 18 to about 25 gauge.
[0102] The invention solid intraocular polymer delivery compositions may
optionally
additionally comprise (e.g., be fabricated to include a means to secure the
solid composition to
ocular tissue at the point of administration, such as a suture tab, to prevent
migration of the
construct to other locations that are not within the intended route of
administration.
(01031 Invention solid polymer intraocular delivery compositions will be
capable of releasing
the ophthalmologic agent over a range from about twenty-four hours, about
seven days, about
thirty days, or about ninety days, or longer, for example up to three years,
depending upon the
condition whose treatment requires release of the ophthalmologic agent. In one
embodiment the
range is from about 1-2 days up to about six months. For example, for delivery
of
ophthalmologic agent to the back of the eye (e.g.., to treat AMD) the
invention solid
composition will release an effective amount of the suitable ophthalmologic
agent over about six
months.
101041 The invention solid compositions can have the ophthalmologic agent
present in a
concentration in the range from about 0.1% up to 99.9% by weight of the
composition. In
addition the invention solid compositions can withstand end stage
sterilization by at least one
method, preferably by multiple methods, e.g., steam, gamma radiation, e-
radiated, and the like.
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[0105] In one embodiment, the invention solid polymer intraocular delivery
compositions can
be physically and chemically stable for a minimum of two years at 5 C,
preferably at 25 C
(room temperature).
[0106] Preferred solid shapes are discs and cylinders; however, any
convenient three
dimensional shape can be used, such as a disc, sheet, film, fiber or tube.
Those of skill in the art
of fluid dynamics will understand that choice of the shape of the solid
composition will also
affect the rate of elution of bioactive agent from the invention composition.
Since, the implant
may be placed during surgery, a cylinder or rectangle sized to fit down the
interior bore of a
hypodermic needle or pharmaceutical delivery needle may be suitable for
placement during the
surgery. In generalõ the invention composition for interior delivery will have
dimensions no
larger than about I mm by 1mm by about 7 mm. For topical application, however,
the size may
be larger. For example, if a sheet is used, the sheet may have any size
suitable for application to
the surface of the eye, for example, about the dimensions of the exterior of
the eye, or about 25
mm x 25 mm.
[0107] In yet further embodiments, the invention solid polymer intraocular
delivery
compositions are porous solids. A "porous solid" fabrication of the invention
polymer
compositions, as the term is used herein, means compositions that have a ratio
of surface area to
volume greater than 1:1. As described below, the maximum porosity of an
invention solid
polymer composition will depend upon its shape and method of fabrication. Any
of the various
methods for creating pores or "scaffolding" for cell growth in polymers may be
used in
connection with the present invention. The following examples of methods for
fabricating the
invention compositions as porous solids are illustrative and not intended to
be limiting.
[0108] In the first example, porosity of the composition is achieved after
the solid polymer
delivery composition is formed by cutting pores through the layers of the
composition, for
example by laser cutting or etching, such as reactive ion etching,. For
example, short-
wavelength UV laser energy is superior to etching for clean-cutting, drilling,
and shaping the
invention polymer composition. UV laser technology first developed by
Massachusetts Institute
of Technology (MIT)allows for removal of very fine and measured amounts of
material as a
plasma plume by "photo-ablation" with each laser pulse, leaving a cleanly-
sculpted pore, or
channel. The large size characteristic of the UV excimer laser beam allows it
to be separated
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into multiple beamlets through near-field imaging techniques, so that multiple
pores, for
example, can be simultaneously bored with each laser pulse. Imaging techniques
also allow sub-
micron resolution so that nano features can be effectively controlled and
shaped. For example,
micro-machining of scaffold thickness of 250 microns and channel depth of 200
microns, with
pore depth of 50 microns has been achieved using this technique on
Polycarbonate, Polyethylene
Terephthalate, and Polyimide.
[0109] In another example, porosity of the invention solid polymer
intraocular delivery
composition is achieved by adding a pore-forming substance, such as a gas, or
a pore-forming
substance (i.e., a porogen) that releases a gas when exposed to heat or
moisture, to the polymer
dispersions and solutions used in casting or spraying the various layers of
the invention delivery
composition,. Such pore-forming substances are well known in the art. For
example,
ammonium bicarbonate salt particles evolve ammonia and carbon dioxide within
the solidifying
polymer matrix upon solvent evaporation. This method results in a product
delivery
composition with layers having vacuoles formed therein by gas bubbles. The
expansion of pores
within the polymer matrix, leads to well interconnected macroporous scaffolds,
for example,
having mean pore diameters of around 300-400 11-m, ideal for high-density in-
growth of cells.
(Y.S. Nam et al., Journal of Biomedical Materials Research Part B: Applied
Biomaterials (2000)
53(1):1-7). Additional techniques known in the art for creating pores in
polymers are the
combination of solvent-casting with particulate-leaching, and temperature-
induced-phase-
separation combined with freeze-drying.
[0110] In yet another embodiment, each layer of the delivery composition is
cast (e.g., spun
by electrospinning) onto the substrate or a preceding layer of the composition
as an
entanglement of fine polymer fibers, such that a polymer mat or pad is formed
upon drying of
the layer. Electrospinning produces polymer fibers with diameter in the range
of 100 nm and
even less, from polymer solutions, suspensions of solid particles and
emulsions by spinning a
droplet in a field of about 1 kV/cm. The electric force results in an
electrically charged jet of
polymer solution out-flowing from a droplet tip. After the jet flows away from
the droplet in a
nearly straight line, the droplet bends into a complex path and other changes
in shape occur,
during which electrical forces stretch and thin the droplet by very large
ratios. After the solvent
evaporates, solidified macro to nanofibers are left (D.H. Reneker et al.
Nanotechnology (1996)
7:216-223).
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101111 The following illustrates estimation of the range of porosity that
can be anticipated for
the above three exemplary modes of fabrication of a rectangular strip of solid
polymer,
wherein SA = surface area of fabrication; V = volume of fabrication; Vp =
volume of
polymer solid; Nv = total number of pores; R = average radius of pores; and
wherein Vv = total volume of pores is defined by Nv*4/38R3 Equation 1
Sv = iota' surface area of pores is defined by 46R2 Equation 2; and
P - porosity is defined by Vv/Vp Equation 3
Porosity of a standard rectangular strip of solid film
101121 For a sphere of polymer of radius R, SAN = 3 per unit length, R.
For a 1 cm3solid block of polymer, SAN = 6 cm-1, i.e. 6 per unit length.
When a 1 cm3 block of solid polymer is pressed into a 1 mm by lcm by 10 cm
strip, the
following holds: SAN = 2.04 or ¨2 mm-1 and V = 1000 mm3 = 1 cm3 (as for the
cube)
So, SA = 22.2 cm2 and SA/V =22.2 cnii. For each type of fabrication described
above, the
surface area to volume ratio of the pores (Sv/Vv) will be calculated in terms
of porosity (P), so
as to
circumvent the necessity of calculating the number of voids, Nv.
Calculations for equivalent, but porous strips or film
(01131 Sv/Vv can be related to SAN most simply when P=1:
For P=1, Vv = Vp = V/2. If the practical approximation that Sv>>SA is made,
then
SA/V = 2SvNp = k*(SvNp) for constant k = 2.
When P>1, in each case SvNp increases linearly with P, and SAN = IcP*(SvNp)
Film with drilled holes (cylindrical pores)
101141 For each hole drilled, the surface lost = 2*i5R2 (on two sides)
For each hole drilled, the surface gained is = 28R*T. Expressed in terms of
porosity, for each
hole drilled, the surface lost = 2*45R2 = 2Vv/T, where T is thickness of the
film.
So, Sv = Nv * [(26RT)-(2Vv/T)] = (2oRT*Vv/oR2".)-(2Vv/T) = 2Vv(1/R - 1/T)
And, Sv/Vv = 2(1/R - 1/T) and SvNp = 2P(1/R - 1/T).
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101151 For P=1, Vv = Vp = V/2 and SvN = 1/R - 1/T= ¨SA/V. Thus, for
2001..im diameter
holes (R = 0.01 cm) drilled through the above standard strip (T = 0.1 cm, V =1
cm3):
SAN = 90 cm -I = 4.5*[(SAN) for solid strip]. Similarly, for holes of half the
above diameter
(R =0.005 cm): SA/V = 190 cml = 9.5*[(SAJV) for solid strip].
101161 For P>1 the integrity of the essentially two dimensional film, for
many applications,
may be compromised. However, the theoretical upper limit of porosity can be
estimated as
follows:
101171 Estimating SAN is linearly proportional to P (from equations above),
for 100 pm
holes, and a porosity of 90%: SAN = 9*9.5*[(SAN) solid strip] = 85.5*[(SAN)
for solid strip];
which suggests an upper limit of SAN ¨100 x.
For a Sponge (formed from gas bubbles)
(0118] Assuming spherical pores of mean radius R: SvNp = Sv*PNv =
3*P*Nv*4612**2/Nv*46R**3 = 3P/R.
101191 If P= l: SvNp = 3/R = ¨ SA/V. If the mean diameter of pores is 200
um (R = 0.01
cm), SvNp = 3*102 = 300 cnril = ¨15*[(SAN) for a solid strip]. Unlike drilling
holes, sponges
can be made with very small pores: If mean diameter of pores is 200 nm (R =
1*10-5 cm),
SvNp = 3*105 cm-I = ¨SAN = 15,000*[(SAN) for solid strip]. Again, SvNp is
linearly
proportional to porosity, P.
101201 If is P>1: For a porosity of 90% , SA/V = 9*15,000*[(SAN) for solid
strip]; which
suggests an upper porosity limit of P ¨150,000x.
For a Fibrous (electro-spun) weave.
101211 The simplest way to approximate an upper limit of SA/V for this mode
of fabrication
is to model a square grid, in which the linear dimension 2R of the cubic pores
is the same as the
thickness of the interleaving polymer sheets. Then, SvNp = 3/2R. Furthermore,
extending this
simplest model to increasing VvNp (i.e. increasing porosity, P), yields SvNp =
3P/2R.
101221 If P=1: For 200 nm diameter fibers (R = 1*10-5 cm), SvNp = 3P//2R =
(3*1)/2*1*10-
s cm or ¨SAN = 7,500*[(SAN) for solid strip].
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101231 If P>1: For a porosity of 90%, SA/V = ¨9*7,500*[(SAN) for solid
strip]; which again
suggests an upper limit of P ¨70,000x.
[01241 In summary then, the broadest envisioned range of surface area over
volume ratios for
drug-eluting films is between 3 and (150,000x20) cm-I; i.e. 3 to 3,000,000
(3M) cm-I.
This range is scaleable; if a very small solid is made with millimeter
dimensions, then SAN can
range from 3 to 3M mm-I. Thus, in general, the upper limit of the range will
be from 3 to 3M
per unit length. That is, the most porous material envisioned could have a
surface area
to volume ratio of up to one million times the equivalent solid sphere.
101251 However, those of skill in the art would understand that, in
practice, the porosity of
the invention polymer solid composition should be considered in light of the
strength
requirements of the particular application for which the composition is
intended, with greater
porosity being suitable for non-weight bearing applications.
101261 Any solid substrate, such as a stainless steel, or a
PolyTetraFluoroEthylene (PTFE)
substrate of any shape, preferably planar, such as a disc, can be used for
casting or spraying of
the various polymer layers that make up the invention solid polymer
intraocular delivery
compositions. For example in one embodiment, the invention composition is
formed as a
sandwich of polymer layers, which are formed by pipetting liquid polymer
solutions or
dispersions onto a stainless steel disc or poly(tetrafluoroethylene)
substrate. The substrate can
be left in place during manufacture of the invention composition and then
removed any time
prior to use.
101271 While the bioactive agents can be dispersed within the polymer
matrix without
chemical linkage to the polymer carrier, it is also contemplated that the
bioactive agent or
covering molecule can be covalently bound to the biodegradable polymers via a
wide variety of
suitable functional groups. For example, when the biodegradable polymer is a
polyester, the
carboxyl group chain end can be used to react with a complimentary moiety on
the bioactive
agent or covering molecule, such as hydroxy, amino, thio, and the like. A wide
variety of
suitable reagents and reaction conditions are disclosed, e.g., in March's
Advanced Organic
Chemistry, Reactions, Mechanisms, and Structure, Fifth Edition, (2001); and
Comprehensive
Organic Transformations, Second Edition, Larock (1999).
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[0128] In other embodiments, a bioactive agent can be linked to the PEA,
PEUR or PEU
polymers described herein through an amide, ester, ether, amino, ketone,
thioether, sulfinyl,
sulfonyl, disulfide linkage. Such a linkage can be formed from suitably
functionalized starting
materials using synthetic procedures that are known in the art.
[0129] For example, in one embodiment a polymer can be linked to the
bioactive agent via an
end or pendent carboxyl group (e.g., COOH) of the polymer. For example, a
compound of
structures IV, VI, and VIII can react with an amino functional group or a
hydroxyl functional
group of a bioactive agent to provide a biodegradable polymer having the
bioactive agent
attached via an amide linkage or carboxylic ester linkage, respectively. In
another embodiment,
the carboxyl group of the polymer can be benzylated or transformed into an
acyl halide, acyl
anhydridermixed" anhydride, or active ester. In other embodiments, the free
¨NH2 ends of the
polymer molecule can be acylated to assure that the bioactive agent will
attach only via a
carboxyl group of the polymer and not to the free ends of the polymer.
[0130] Water soluble covering molecule(s), such as poly(ethylene glycol)
(PEG); phosphoryl
choline (PC); glycosaminoglycans including heparin; polysaccharides including
polysialic acid;
poly(ionizable or polar amino acids) including polyserine, polyglutamic acid,
polyaspartic acid,
polylysine and polyarginine; chitosan and alginate, as described herein, and
targeting molecules,
such as antibodies, antigens and ligands, can also be conjugated to the
polymer in the exterior of
the particles after production of the particles to block active sites not
occupied by the bioactive
agent or to target delivery of the particles to a specific body site as is
known in the art. The
molecular weights of PEG molecules on a single particle can be substantially
any molecular
weight in the range from about 200 to about 200,000, so that the molecular
weights of the
various PEG molecules attached to the particle can be varied.
[0131] Alternatively, the bioactive agent or covering molecule can be
attached to the polymer
via a linker molecule, for example, as described in structural formulas (XI,
XII). Indeed, to
improve surface hydrophobicity of the biodegradable polymer, to improve
accessibility of the
biodegradable polymer towards enzyme activation, and to improve the release
profile of the
biodegradable polymer, a linker may be utilized to indirectly attach the
bioactive agent to the
biodegradable polymer. In certain embodiments, the linker compounds include
poly(ethylene
glycol) having a molecular weight (MW) of about 44 to about 10,000, preferably
44 to 2000;
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amino acids, such as serine; polypeptides with repeat number from 1 to 100;
and any other
suitable low molecular weight polymers. The linker typically separates the
bioactive agent from
the polymer by about 5 angstroms up to about 200 angstroms.
[01321 In still further embodiments, the linker is a divalent
radical of formula W-A-Q,
wherein A is (C1-C24) alkyl, (C2-C24) alkenyl, (C2-C24) alkynyl, (C3-C8)
cycloalkyl, or (C6-Cio)
aryl, and W and Q are each independently ¨N(R)C(=0)-, -C(=0)N(R)-, -0C(=0)-, -
C(=0)0, -
0-, -S-, -S(0), -S(0)2-, -S-S-, -N(R)-, -C(=0)-, wherein each R is
independently H or (Ci-C6)
alkyl.
101331 As used to describe the above linkers, the term "alkyl"
refers to a straight or branched
chain hydrocarbon group including methyl, ethyl, n-propyl, isopropyl, n-butyl,
isobutyl, tert-
,
butyl, n-hexyl, and the like.
101341 As used herein to refer to a linker, "alkenyl" refers to
straight or branched chain
hydrocarbyl groups having one or more carbon-carbon double bonds.
101351 As used herein to refer to a linker, "alkynyl" refers to
straight or branched chain
hydrocarbyl groups having at least one carbon-carbon triple bond.
101361 As used herein to refer to a linker, "aryl" refers to
aromatic groups having in the range
of 6 up to 14 carbon atoms.
[01371 In certain embodiments, the linker may be a polypeptide
having from about 2 up to
about 25 amino acids. Suitable peptides contemplated for use include poly-L-
glycine, poly-L-
lysine, poly-L-glutamic acid, poly-L-aspartic acid, poly-L-histidine, poly-L-
ornithine, poly-L-
serine, poly-L-threonine, poly-L-tyrosine, poly-L-leucine, poly-L-lysine-L-
phenylalanine, poly-
L-arginine, poly-L-lysine-L-tyrosine, and the like.
[0138] In one embodiment, the bioactive agent can covalently
crosslink the polymer, i.e. the
bioactive agent is bound to more than one polymer molecule. This covalent
crosslinking can be
done with or without additional polymer-bioactive agent linker.
[01391 The bioactive agent molecule can also be incorporated into an
intramolecular bridge
by covalent attachment between two polymer molecules.
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101401 A linear polymer polypeptide conjugate is made by protecting the
potential
nucleophiles on the polypeptide backbone and leaving only one reactive group
to be bound to
the polymer or polymer linker construct. Deprotection is performed according
to methods well
known in the art for deprotection of peptides (Boc and Fmoc chemistry for
example).
101411 In one embodiment of the present invention, a polypeptide bioactive
agent is
presented as retro-inverso or partial retro-inverso peptide.
101421 In other embodiments the bioactive agent is mixed with a
photocrosslinkable version
of the polymer in a matrix, and after crosslinking the material is dispersed
(ground) to an
average diameter in the range from about 0.1 to about 10 m.
101431 The linker can be attached first to the polymer or to the bioactive
agent or covering
molecule. During synthesis, the linker can be either in unprotected form or
protected form,
using a variety of protecting groups well known to those skilled in the art.
In the case of a
protected linker, the unprotected end of the linker can first be attached to
the polymer or the
bioactive agent or covering molecule. The protecting group can then be de-
protected using
Pd/I-12 hydrogenolysis, mild acid or base hydrolysis, or any other common de-
protection method
that is known in the art. The de-protected linker can then be attached to the
bioactive agent or
covering molecule, or to the polymer
[01441 An exemplary synthesis of a biodegradable polymer according to the
invention
(wherein the molecule to be attached is an aminoxyl) is set forth as follows.
[01451 A polyester can be reacted with an amino-substituted aminoxyl (N-
oxide) radical
bearing group, e.g., 4-amino-2,2,6,6-tetramethylpiperidine-1-oxy, in the
presence of N,N'-
carbonyldiimidazole to replace the hydroxyl moiety in the carboxyl group at
the chain end of the
polyester with an amino-substituted aminoxyl-(N-oxide) radical bearing group,
so that the amino
moiety covalently bonds to the carbon of the carbonyl residue of the carboxyl
group to form an
amide bond. The N,N'-carbonyl diimidazole or suitable carbodiimide converts
the hydroxyl
moiety in the carboxyl group at the chain end of the polyester into an
intermediate product
moiety which will react with the aminoxyl, e.g., 4-amino-2,2,6,6-
tetramethylpiperidine-1-oxy.
The aminoxyl reactant is typically used in a mole ratio of reactant to
polyester ranging from 1:1
to 100: I. The mole ratio of N,N'-carbonyl diimidazole to aminoxyl is
preferably about 1:1.
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[01461 A typical reaction is as follows. A polyester is dissolved in a
reaction solvent and
reaction is readily carried out at the temperature utilized for the
dissolving. The reaction solvent
may be any in which the polyester will dissolve. When the polyester is a
polyglycolic acid or a
poly(glycolide-L-lactide) (having a monomer mole ratio of glycolic acid to L-
lactic acid greater
than 50:50), highly refined (99.9+% pure) dimethyl sulfoxide at 115 C to 130
C or DMSO at
room temperature suitably dissolves the polyester. When the polyester is a
poly-L-lactic acid, a
poly-DL-lactic acid or a poly(glycol ide-L-lactide) (having a monomer mole
ratio of glycolic acid
to L-lactic acid 50:50 or less than 50:50), tetrahydrofuran, dichloromethane
(DCM) and
chloroform at room temperature to 40 ¨50 C suitably dissolve the polyester.
Polymer - Bioactive agent Linkage
101471 In one embodiment, the polymers used to make the invention polymer
particle
delivery compositions as described herein have one or more bioactive agent
directly linked to the
polymer. The residues of the polymer can be linked to the residues of the one
or more bioactive
agents. For example, one residue of the polymer can be directly linked to one
residue of the
bioactive agent. The polymer and the bioactive agent can each have one open
valence.
Alternatively, more than one bioactive agent, multiple bioactive agents, or a
mixture of bioactive
agents having different therapeutic or palliative activity can be directly
linked to the polymer.
However, since the residue of each bioactive agent can be linked to a
corresponding residue of
the polymer, the number of residues of the one or more bioactive agents can
correspond to the
number of open valences on the residue of the polymer.
101481 As used herein, a "residue of a polymer" refers to a radical of a
polymer having one or
more open valences. Any synthetically feasible atom, atoms, or functional
group of the polymer
(e.g., on the polymer backbone or pendant group) of the present invention can
be removed to
provide the open valence, provided bioactivity is substantially retained when
the radical is
attached to a residue of a bioactive agent. Additionally, any synthetically
feasible functional
group (e.g., carboxyl) can be created on the polymer (e.g., on the polymer
backbone or pendant
group) to provide the open valence, provided bioactivity is substantially
retained when the
radical is attached to a residue of a bioactive agent. Based on the linkage
that is desired, those
skilled in the art can select suitably functionalized starting materials that
can be derived from the
polymer of the present invention using procedures that are known in the art.
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[0149] As used herein, a "residue of a compound of structural formula (*)"
refers to a radical
of a compound of polymer formulas (I) and (IV-VIII) as described herein having
one or more
open valences. Any synthetically feasible atom, atoms, or functional group of
the compound
(e.g., on the polymer backbone or pendant group) can be removed to provide the
open valence,
provided bioactivity is substantially retained when the radical is attached to
a residue of a
bioactive agent. Additionally, any synthetically feasible functional group
(e.g., carboxyl) can be
created on the compound of formulas (I) and (IV-VIII) (e.g., on the polymer
backbone or
pendant group) to provide the open valance, provided bioactivity is
substantially retained when
the radical is attached to a residue of a bioactive agent. Based on the
linkage that is desired,
those skilled in the art can select suitably functionalized starting materials
that can be derived
from the compound of formulas (I) and (IV¨VIII) using procedures that are
known in the art.
[01501 For example, the residue of a bioactive agent can be linked to the
residue of a
compound of structural formula (I) or (IV-VIII) through an amide (e.g., -
N(R)C(=0)- or
¨C(=0)N(R)-), ester (e.g., -0C(=0)- or ¨C(=0)0-), ether (e.g., -0-), amino
(e.g., -N(R)-),
ketone (e.g., -C(=0)-), thioether (e.g., -S-), sulfinyl (e.g., -S(0)-),
sulfonyl (e.g., -S(0)2-),
disulfide (e.g., -S-S-), or a direct (e.g., C-C bond) linkage, wherein each R
is independently H or
(CI-C6) alkyl. Such a linkage can be formed from suitably functionalized
starting materials
using synthetic procedures that are known in the art. Based on the linkage
that is desired, those
skilled in the art can select suitably functional starting material that can
be derived from a
residue of a compound of structural formula (I) or (IV-VIII) and from a given
residue of a
bioactive agent or adjuvant using procedures that are known in the art. The
residue of the
bioactive agent or adjuvant can be linked to any synthetically feasible
position on the residue of
a compound of structural formula (I) or (IV). Additionally, the invention also
provides
compounds having more than one residue of a bioactive agent or adjuvant
bioactive agent
directly linked to a compound of structural formula (I) or (IV).
[0151] The number of bioactive agents that can be linked to the polymer
molecule can
typically depend upon the molecular weight of the polymer. For example, for a
compound of
structural formula (1), wherein n is about 5 to about 150, preferably about 5
to about 70, up to
about 150 bioactive agent molecules (i.e., residues thereof) can be directly
linked to the polymer
(i.e., residue thereof) by reacting the bioactive agent with side groups of
the polymer. In
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unsaturated polymers, the bioactive agents can also be reacted with double (or
triple) bonds in
the polymer.
101521 The number of bioactive agents that can be linked to the polymer
molecule can
typically depend upon the molecular weight of the polymer. For example, for a
saturated
compound of structural formula (I), wherein n is about 5 to about 150,
preferably about 5 to
about 70, up to about 150 bioactive agents (i.e., residues thereof) can be
directly linked to the
polymer (i.e., residue thereof) by reacting the bioactive agent with side
groups of the polymer.
In unsaturated polymers, the bioactive agents can also be reacted with double
(or triple) bonds in
the polymer.
101531 In one embodiment, the PEA, PEUR and PEU polymers, and mixtures or
blends
thereof, can be used to formulate biodegradable polymer particle intraocular
delivery
compositions for time release of at least one ophthalmologic agent in a
consistent and reliable
manner. These polymer particle delivery compositions can also incorporate an
ophthalmologic
diol, or a residue of a therapeutic diol or di-acid) into the backbone of the
polymer for time
release of the bioactive agent from the backbone of the polymer in a
consistent and reliable
manner by biodegradation of the polymers in the polymer particles.
[01541 PEA, PEUR and PEU polymers described herein absorb water, (5 to 25 %
w/w water
up-take, on polymer film) allowing hydrophilic molecules to readily diffuse
therethrough. This
characteristic makes these polymers suitable for use as an over coating on
particles to control
release rate. Water absorption also enhances biocompatibility of the polymers
and the polymer
particle delivery composition based on such polymers. In addition, due to the
hydrophilic
properties of the PEA, PEUR and PEU polymers, when delivered intraocularly the
particles
become sticky and agglomerate, particularly at in vivo temperatures. Thus the
polymer particles
spontaneously form polymer depots when injected intraocularly for local
delivery. Particles
with average diameter range from about 1 micron to about 100 microns, which
size will not
circulate efficiently within the body, are suitable for forming such polymer
depots intraocularly.
(01551 Methods of making intraocular polymer particle delivery compositions
Particles
suitable for use in the invention intraocular polymer delivery composition can
be made using
immiscible solvent techniques and as described herein and in copending U.S.
applications
60/684,670, filed May 25, 2005; 60/737,401, filed November 14, 2005;
60/687,570, filed June 3,
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2005; 60/759,179, filed January 13, 2006 (Atty Docket MEDIV2070-1);
60/719,950, filed
September 22, 2005. Generally,
these methods entail the preparation of an emulsion of two immiscible liquids.
A single
emulsion method can be used to make polymer particles that incorporate at
least one
hydrophobic bioactive agent. In the single emulsion method, bioactive agents
to be incorporated
into the particles are mixed with polymer in solvent first, and then
emulsified in water solution
with a surface stabilizer, such as a surfactant. In this way, polymer
particles with hydrophobic
6ioactive agent conjugates are formed and suspended in the water solution, in
which
hydrophobic conjugates in the particles will be stable without significant
elution into the
aqueous solution, but such molecules will elute into body tissue, such as
muscle tissue.
101561 Most biologics, including polypeptides, proteins, DNA, cells and
the like, are
hydrophilic. A double emulsion method can be used to make polymer particles
with interior
aqueous phase and hydrophilic bioactive agents dispersed within. In the double
emulsion
method, aqueous phase or hydrophilic bioactive agents dissolved in water are
emulsified in
polymer lipophilic solution first to form a primary emulsion, and then the
primary emulsion is
put into water to emulsify again to form a second emulsion, in which particles
are formed having
a continuous polymer phase and aqueous bioactive agent(s) in the dispersed
phase. Surfactant
and additive can be used in both emulsifications to prevent particle
aggregation. Chloroform or
DCM, which are not miscible in water, are used as solvents for PEA and PEUR
polymers, but
later in the preparation the solvent is removed, using methods known in the
art.
101571 For certain bioactive agents with low water solubility, however,
these two emulsion
methods have limitations. In this context, "low water solubility" means a
bioactive agent that is
less hydrophobic than truly lipophilic drugs, such as Taxol, but which are
less hydrophilic than
truly water-soluble drugs, such as many biologics. These types of intermediate
compounds are
too hydrophilic for high loading and stable matrixing into single emulsion
particles, yet are too
hydrophobic for high loading and stability within double emulsions. In such
cases, a polymer =
layer is coated onto particles made of polymer and drugs with low water
solubility, by a triple
emulsion process. This method provides relatively low drug loading (-10% w/w),
but provides
structure stability and controlled drug release rate.
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101581 In the triple emulsion process, the first emulsion is made by
mixing the bioactive
agents into polymer solution and then emulsifying the mixture in aqueous
solution with
surfactant or lipid, such as di-(hexadecanoyl) phosphatidylcholine (DHPC; a
short-chain
derivative of a natural lipid). In this way, particles containing the active
agents are formed and
suspended in water to form the first emulsion. The second emulsion is formed
by putting the
first emulsion into a polymer solution, and emulsifying the mixture, so that
water drops with the
polymer/drug particles inside are formed within the polymer solution. Water
and surfactant or
lipid will separate the particles and dissolve the particles in the polymer
solution. The third
emulsion is then formed by putting the second emulsion into water with
surfactant or lipid, and
emulsifying the mixture to form the final particles in water. The resulting
particle structure will
have one or more particles made with polymer plus bioactive agent at the
center, surrounded by
water and surface stabilizer, such as surfactant or lipid, and covered with a
pure polymer shell.
Surface stabilizer and water will prevent solvent in the polymer coating from
contacting the
particles inside the coating and dissolving them.
[01591 To increase loading of bioactive agents by the triple emulsion
method, active agents
with low water solubility can be coated with surface stabilizer in the first
emulsion, without
polymer coating and without dissolving the bioactive agent in water. In this
first emulsion,
water, surface stabilizer and active agent have similar volume or in the
volume ratio range of (1
to 3):(0.2 to about 2):1, respectively. In this case, water is used, not for
dissolving the active
agent, but rather for protecting the bioactive agent with help of surface
stabilizer. Then the
double and triple emulsions are prepared as described above. This method can
provide up to
50% drug loading.
(0160) Alternatively or additionally in the single, double or triple
emulsion methods
described above, a bioactive agent can be conjugated to the polymer molecule
as described
herein prior to using the polymers to make the particles. Alternatively still,
a bioactive agent can
be conjugated to the polymer on the exterior of the particles described herein
after production of
the particles.
(0161) Many emulsification techniques will work in making the emulsions
described above.
However, the presently preferred method of making the emulsion is by using a
solvent that is not
= miscible in water. For example, in the single emulsion method, the
emulsifying procedure
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consists of dissolving polymer with the solvent, mixing with bioactive agent
molecule(s), putting
into water, and then stirring with a mixer and/or ultra-sonicator. Particle
size can be controlled
by controlling stir speed and/or the concentration of polymer, bioactive
agent(s), and surface
stabilizer. Coating thickness can be controlled by adjusting the ratio of the
second to the third
emulsion.
101621 Suitable emulsion stabilizers may include nonionic surface active
agents, such as
mannide monooleate, dextran 70,000, polyoxyethylene ethers, polyglycol ethers,
and the like, all
readily commercially available from, e.g., Sigma Chemical Co., St. Louis, Mo.
The surface
active agent will be present at a concentration of about 0.3% to about 10%,
preferably about
0.5% to about 8%, and more preferably about I% to about 5%.
101631 Rate of release of the at least one bioactive agent from the
invention compositions can
be controlled by adjusting the coating thickness, particle size, structure,
and density of the
coating. Density of the coating can be adjusted by adjusting loading of the
bioactive agent
conjugated to the coating. For example, when the coating contains no bioactive
agent, the
polymer coating is densest, and a bioactive agent from the interior of the
particle elutes through
the coating most slowly. By contrast, when a bioactive agent is loaded into
(i.e. is mixed or
"matrixed" with), or alternatively is conjugated to, polymer in the coating,
the coating becomes
porous once the bioactive agent has become free of polymer and has eluted out,
starting from the
outer surface of the coating. Thereby, a bioactive agent at the center of the
particle can elute at
an increased rate. The higher the bioactive agent loading in the coating, the
lower the density of
the coating layer and the higher the elution rate. The loading of bioactive
agent in the coating
can be lower or higher than that in the interior of the particles beneath the
exterior coating.
Release rate of bioactive agent(s) from the particles can also be controlled
by mixing particles
with different release rates prepared as described above.
101641 A detailed description of methods of making double and triple
emulsion polymers
may be found in Pierre Autant et al, Medicinal and/or nutritional
microcapsules for oral
administration, U.S. patent No. 6,022,562; losif Daniel Rosca et al.,
Microparticle formation and
its mechanism in single and double emulsion solvent evaporation, Journal of
Controlled Release
99 (2004) 271-280; L. Mu, S.S. Feng, A novel controlled release formulation
for the anticancer
drug paclitaxel (Taxol): PLGA nanoparticles containing vitamin E TPGS, J.
Control. Release 86
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52
(2003) 33¨ 48; Somatosin containing biodegradable microspheres prepared by a
modified
solvent evaporation method based on W/O/VV-multiple emulsions, Int. J. Pharm.
126 (1995)
129¨ 138 and F. Gabor, 13. Ertl, M. Wirth, R. Ma!linger, Ketoprofenpoly(d,l-
lactic-co-glycolic
acid) microspheres: influence of manufacturing parameters and type of polymer
on the release
characteristics, J. Microencapsul. 16 (1) (1999) 1¨ 12.
101651 In yet further embodiments for delivery of aqueous-soluble
bioactive agents, the
particles can be made into nanoparticles having an average diameter of about
20 nm to about 200
nm for delivery to the circulation. The nanoparticles can be made by the
single emulsion
method with the active agent dispersed therein, i.e., mixed into the emulsion
or conjugated to
polymer as described herein. The nanoparticles can also be provided as a
micellar composition
containing the polymers described herein, such as PEA and PEUR with the
bioactive agents
conjugated thereto. Alternatively or in addition to bioactive agents
conjugated to the polymers,
since the micelles are formed in water, water soluble bioactive agents can be
loaded into the
micelles at the same time without solvent.
101661 More particularly, the biodegradable micelles are formed of a
hydrophobic polymer
chain conjugated to a water soluble polymer chain. Whereas, the outer portion
of the micelle
mainly consists of the water soluble ionized or polar section of the polymer,
the hydrophobic
section of the polymer mainly partitions to the interior of the micelles and
holds the polymer
molecules together.
101671 The biodegradable hydrophobic section of the polymer used to make
micelles is made
of PEA, PEUR or PEU polymers, as described herein. For strongly hydrophobic
PEA, PEUR or
PEU polymers, components such as di-- L-leucine ester of 1,4:3,6-dianhydro-D-
sorbitol or rigid
aromatic di-acid like a,to-bis (4-carboxyphenoxy) (C1-C8) alkane may be
included in the
polymer repeat unit. By contrast, the water soluble section of the polymer
comprises repeating
alternating units of polyethylene glycol, polyglycosaminoglycan or
polysaccharide and at least
one ionizable or polar amino acid, wherein the repeating alternating units
have substantially
similar molecular weights and wherein the molecular weight of the polymer is
in the range from
about 10 kDa to about 300 IcDa. The repeating alternating units may have
substantially similar
molecular weights in the range from about 300 Da to about 700 Da. In one
embodiment wherein
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the molecular weight of the polymer is over 10 kDa, at least one of the amino
acid units is an
ionizable or polar amino acid selected from serine, glutamic acid, aspartic
acid, lysine and
arginine. In one embodiment, the units of ionizable amino acids comprise at
least one block of
ionizable poly(amino acids), such as glutamate or aspartate, can be included
in the polymer. The
invention micellar composition may further comprise a pharmaceutically
acceptable aqueous
media with a pH value at which at least a portion of the ionizable amino acids
in the water
soluble sections of the polymer are ionized.
101681 The higher the molecular weight of the water soluble section of the
polymer, the
greater the porosity of the micelle and the higher the loading into the
micelles of water soluble
bioactive agents ancUor large bioactive agents, such as proteins. In one
embodiment, therefore,
the molecular weight of the complete water soluble section of the polymer is
in the range from
about 5 kDa to about 100 kDa.
[01691 Once formed, the micelles can be lyophilized for storage and
shipping and
reconstituted in the above-described aqueous media. However, it is not
recommended to
lyophilize micelles containing certain bioactive agents, such as certain
proteins, that would be
denatured by the lyophilization process.
[0170] Charged moieties within the micelles partially separate from each
other in water, and
create space for absorption of water soluble agents, such as the bioactive
agent(s). Ionized
chains with the same type of charge will repel each other and create more
space. The ionized
polymer also attracts the bioactive agent, providing stability to the matrix.
In addition, the water
soluble exterior of the micelle prevents adhesion of the micelles to proteins
in body fluids after
ionized sites are taken by the therapeutic bioactive agent. This type of
micelle has very high
.porosity, up to 95% of the micelle volume, allowing for high loading of
aqueous-soluble
biologics, such as polypeptides, DNA, and other bioactive agents. Particle
size range of the
micelles is about 20 nm to about 200nm, with about 20 nm to about 100 nm being
preferred for
circulation in the blood.
[01711! Particle size can be determined by, e.g., laser light scattering,
using for example, a
spectrometer incorporating a helium-neon laser. Generally, particle size is
determined at room
temperature and involves multiple analyses of the sample in question (e.g., 5-
10 times) to yield
an average value for the particle diameter. Particle size is also readily
determined using
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scanning electron microscopy (SEM). In order to do so, dry particles are
sputter-coated with a
gold/palladium mixture to a thickness of approximately 100 Angstroms, and then
examined
using a scanning electron microscope. Alternatively, the polymer, either in
the form of particles
or not, can be covalently attached directly to the bioactive agent, rather
than incorporating active
agent therein ("loading) without chemical attachment, using any of several
methods well known
in the art and as described hereinbelow. The bioactive agent content is
generally in an amount
that represents approximately 0.1% to about 40% (w/w) bioactive agent to
polymer, more
preferably about 1% to about 25% (w/w) bioactive agent, and even more
preferably about 2% to
about 20% (w/w) bioactive agent. The percentage of bioactive agent will depend
on the desired
dose and the condition being treated, as discussed in more detail below.
[0172) Methods of making intraocular solid polymer delivery compositions To
fabricate the
invention intraocular solid polymer delivery composition for controlled
release of an
ophthalmologic agent, the following polymer layers can be cast or sprayed onto
a solid substrate:
a) at least one carrier layer comprising a liquid solution in a first
solvent of at least
one bioactive agent and a biodegradable, biocompatible polymer having a
structural formula
described by structural formula (I) - (IV-V1II) as described herein;
b) at least one coating layer of a liquid solution in a second solvent of a
biodegradable, biocompatible polymer; and
c) at least one barrier layer of a liquid polymer that is insoluble in the
second
solvent, but dissolves under physiological conditions, wherein the barrier
layer is between the
carrier layer and each coating layer. Each layer is dried before casting or
spraying the next layer
thereon. A polymer layer cast or sprayed onto the substrate as a liquid
dispersion forms a film
(for example, a substantially planar body having opposed major surfaces and a
thickness
between the major surfaces of from about 0.1 millimeters to about 20
millimeters, e.g. 5
millimeters).
101731 Alternatively, for constructs having a thickness of about 0.1. to
2.5 mm in thickness, a
carrier layer comprising a liquid solution in a first solvent of at least one
biodegradable,
biocompatible polymer with dispersed ophthalmologic agent is sprayed onto the
solid substrate
and dried. Then a single coating layer of a liquid solution of a
biodegradable, biocompatible
polymer in the same solvent or in a second solvent is sprayed atop the carrier
layer and dried.
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101741 Drying of the various layers can be accomplished using any method
known in the art
so long as the temperature is not high enough to breakdown the chemical
structure of the various
polymers used or that of the one or more bioactive agents dispersed in the
carrier layer.
Typically, temperatures do not exceed 80 C. For example, the various layers
of the composition
can be dried in an oven at a temperature of 40 C to about 50 C for a period
of about 5 hours to
= about 9 hours, for example, about 7 hours at 50 C, as is illustrated in
the Examples herein.
(01751 In one embodiment, the coating layer(s) as well as the carrier
layer are applied using a
liquid solution of a polymer having a chemical formula described by structural
formulas (I) and
(IV). Although in no way necessary for practice of the invention, for
convenience, it is
recommended to use the same combination of solvent and biodegradable,
biocompatible
polymer of structures (I) - (IV) in the coating layer(s) as in the carrier
layer(s) of the invention
composition. For example, the polymer used in casting or spraying the coating
layer(s) can be
the same as that used in casting or spraying the carrier layer(s), in which
case the first solvent
and the second solvent can be identical.
101761 The composition of the barrier layer(s) in the invention solid
polymer intraocular
delivery composition is an important aspect of the invention. The choice of
the polymer used in
the barrier layer(s) is determined by the solvent used to form the solution
for preparation of the
covering layer(s). The purpose of the barrier layer(s) being to prevent
uncontrolled solvation of
the bioactive agent out of the carrier layer(s) during deposition of the
covering layer with which
it would otherwise be in contact. The polymer for the intermediate barrier
layer(s) is selected to
be insoluble in solvent used in formation of the covering layer(s). In one
embodiment, the
barrier layer is a monolayer of polymer in the finished composition. In
addition, all polymers
used in the various layers of the solid polymer delivery composition are
biocompatible and will
be re-absorbed by the body through natural enzymatic action. In another
embodiment, the
coating layer(s) applied are free of bioactive agents and may be referred to
herein as a "pure
polymer layer".
[01771 For example, a polymer that dissolves in ethanol, such as copolymer
PEA (a form of
the polymer represented in formula (IV)), can be used in a solution of this
solvent in applying
the carrier and coating layers, and a polyvinyl alcohol, which is insoluble in
ethanol, can be used
to make one or more barrier layers in the composition. In one embodiment, the
liquid polymer
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that forms the barrier layer(s) is applied so as to form a monolayer of the
polymer, e.g.,
polyvinyl alcohol.
101781 To further prolong the release period from the invention solid
polymer intraocular
delivery composition, the composition has a three-dimensional sandwich
structure. The method
of making the composition may then be modified by first casting multiple sets
of the coating
layer and barrier layer onto the substrate, beginning with a coating layer
(drying each before
deposit of the next) prior to casting the carrier layer. Then, to form the
interior of the sandwich
structure, the carrier layer is cast and dried. Finally, in reverse order,
multiple sets of the barrier
layer and coating layer are cast atop the dried barrier layer. In this way, a
coating layer is first
and last to be deposited and the composition takes on a sandwich structure,
wherein the multiple
sets are applied twice to form two external sides of a three-dimensional
sandwich structure, with
a coating layer being external to both of the two sides of the structure and
with the carrier layer
being at the center of the sandwich structure. Each composition construct,
therefore, may
contain no set of the layers or about one to about ten barrier and coating
layer sets (e.g., 1 to
about 8 sets of barrier and coating layers).
(0179) In a more schematic format, for this embodiment the following
procedure can be
followed to make a composition having N sets of coating and barrier layers in
a sandwich
structure, with the carrier layer at the center of the sandwich structure:
1. Cast/spray Nth layer of coating onto substrate and dry,
2. Coat with Nth barrier layer and dry,
3. Cast/spray (N-1) th coating layer and dry,
4. Coat with (N-1) th barrier layer and dry,
5. Repeat steps 3 and 4 as desired.
6. Cast/spray carrier layer containing one or more matrixed bioactive agent
(the
carrier layer will end up as the inner-most layer of the sandwich) and dry.
7. Repeat steps 5 to 1 in the reverse order, ending with a coating layer.
Preferably
each liquid layer deposited will over run the edge of the previous one to seal
the sides of the
composition being formed so that elution from the sides of the disc will be
controlled as well as
from other portions of the surface area. The substrate can be removed from the
dried layers at
any point in the method of making the invention composition afier one or two
layers have been
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cast and dried thereon. The completed composition can be packaged for storage
or is ready for
immediate use.
[0180] Alternatively, if the solvent is not completely removed from the
various layers of the
composition during the drying steps described above, the composition can be
manipulated and
compressed to form any of a number of three dimensional shapes, such as by
rolling, pleating,
folding, and the like, prior to a final drying to substantially remove solvent
from the
composition. For example, a disc can be rolled up and compressed to form a
cylinder.
Alternatively, cylinders can be punched with a dye from a sheet of fully dried
material formed
layer by layer according to the above described methods.
(01811 For thinner composition constructs, primarily where the total
thickness of the
construct does not exceed approximately 2.5 mm, an aerosol of solvated polymer
matrixed with
bioactive molecule(s) is deposited on a substrate. Alternatively, the carrier
layer can be solution
cast. This carrier layer is then dried by methodology outlined in c). For the
deposition of the
coating layer(s) using an aerosol, diffusion of the drug or biologic out of
the carrier layer and
into the wet coating layer being formed is limited by three factors: 1)
concentration of the
biodegradable, biocompatible polymer solution is typically as high as possible
(c.a. 2% to 3%)
without forming solids in the air, such that the aerosol is nearly dry upon
deposition on the
surface, 2) substrate and/or airspace above are continuously heated to promote
rapid drying
(typically 30 to 80 ''C, depending on the polymer and concentration), and 3) a
single coat is
divided into many "spray cycles." By using "spray cycles," the period of
deposition of the
aerosol is divided into many shorter periods of less than a second in length
to several seconds.
Each spray period is followed by a short drying period (e.g. 20 to 60 seconds,
incorporating
continuous heating in each cycle), such that drying is rapid and residual
solvent is minimized for
a given coating. Heat can typically be applied continuously, even during the
aerosol deposition
periods of the spray cycle. Drying of the newly formed coating layer is
performed as described
herein. Any single coat typically does not exceed approximately 80 pm.
Although migration of
the biologic ancUor drug into the newly formed coating layer may not be
completely eliminated
as in embodiments employing the barrier layer, migration is limited to the
extent that diffusion
of small molecules in the final product is slower than in those embodiments
containing only a
carrier layer. The rate of release of the ophthalmologic agent and optional
bioactive molecules is
thereby controlled.
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70500-195
58
101821 Methods of making solid polymer implantables containing PEA, PEUR and
PEU
polymers are further disclosed in U. S. application 11/525,491, filed
September 21 2006.
(01831 Bioactive agents for optional dispersion into and release from
the invention
biodegradable intraocular polymer delivery compositions also include anti-
proliferants,
rapamycin and any of its analogs or derivatives, paclitaxel or any of its
taxene analogs or
derivatives, everolimus, Sirolimus, tacrolimus, or any of its ¨limus named
family of drugs, and
statins such as simvastatin, atorvastatin, fluvastatin, pravastatin,
lovastatin, rosuvastatin,
geldanamycins, such as I 7AAG (I 7-allyiamino-17-demethoxygeldanamycin);
Epothilone D and
other epothilones, 17-dimethylaminoethylamino-17-demethoxy-geldanamycin and
other
polyketide inhibitors of heat shock protein 90 (Hsp90), Cilostazol, and the
like.
(0184j The following bioactive agents and small molecule drugs can
optionally be dispersed
within the invention intraocular delivery compositions, whether formulated and
sized to form a
time release biodegradable polymer depot for local delivery of the bioactive
agents or fabricated
as solid delivery systems. Any optional bioactive agents that are dispersed in
the polymers used
in the invention delivery compositions and methods of delivery will be
selected for their suitable
therapeutic or palliative effect in treatment of an ophthalmologic disease of
interest, or
symptoms thereof.
(01851 In one embodiment, the optional bioactive agents are not limited
to, but include,
various classes of compounds that facilitate or contribute to wound healing
when presented in a
time-release fashion.
101861 Small molecule drugs are a category of additional bioactive
agents suitable for
dispersion in the invention intraocular polymer delivery compositions
described herein. Such
drugs include, for example, antimicrobials and anti-inflammatory agents as
well as certain
healing promoters, such as, for example, vitamin A and synthetic inhibitors of
lipid peroxidation.
101871 A variety of antibiotics can be dispersed in the invention
intraocular polymer delivery
compositions to indirectly promote natural healing processes by preventing or
controlling
infection. Suitable antibiotics include many classes, such as arninoglycoside
antibiotics or
quinolones or beta-lactams, such as cefalosporins, e.g., ciprofloxacin,
gentamycin, tobramycin,
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erythromycin, vancomycin, oxacillin, cloxacillin, methicillin, lincomycin,
ampicillin, and
colistin. Suitable antibiotics have been described in the literature.
10188] Suitable antimicrobials include, for example, Adriamycin PFS/RDFO
(Pharmacia and
Upjohn), Blenoxane0 (Bristol-Myers Squibb Oncology/Immunology), Cerubidine0
(Bedford),
Cosmegen0 (Merck), DaunoXomee (NeXstar), Doxile (Sequus), Doxorubicin
Hydrochloride (Astra), IdamycindPFS (Pharmacia and Upjohn), Mithracin
(Bayer),
Mitamycine (Bristol-Myers Squibb Oncology/Immunology), Nipene (SuperGen),
Novantrone (1mmunex) and Rubex0 (Bristol-Myers Squibb Oncology/Immunology).
In one
embodiment, the peptide can be a glycopeptide. "Glycopeptide" refers to
oligopeptide (e.g.
heptapeptide) antibiotics, characterized by a multi-ring peptide core
optionally substituted with
saccharide groups, such as vancomycin.
101891 Examples of glycopeptides included in this category of
antimicrobials may be found
in "Glycopeptides Classification, Occurrence, and Discovery," by Raymond C.
Rao and Louise
W. Crandall, ("Bioactive agents and the Pharmaceutical Sciences" Volume 63,
edited by
Ramakrishnan Nagarajan, published by Marcal Dekker, Inc.). Additional examples
of
glycopeptides are disclosed in U.S. Patent Nos. 4,639,433; 4,643,987;
4,497,802; 4,698,327,
5,591,714; 5,840,684; and 5,843,889; in EP 0 802 199; EP 0 801 075; EP 0 667
353; WO
97/28812; WO 97/38702; WO 98/52589; WO 98/52592; and in J. Amer. Chem. Soc.,
1996, 118,
13107-13108; J. Amer. Chem. Soc., 1997, 119, 12041-12047; and J. Amer. Chem.
Soc., 1994,
116, 4573-4590. Representative glycopeptides include those identified as A477,
A35512,
A40926, A41030, A42867, A47934, A80407, A82846, A83850, A84575, AB-65,
Actaplanin,
Actinoidin, Ardacin, Avoparcin, Azureomycin, Balhimyein, Chloroorientiein,
Chloropolysporin,
Decaplanin, -demethylvancomycin, Eremomycin, Galacardin, Helvecardin,
Izupeptin, Kibdelin,
LL-AM374, Mannopeptin, MM45289, MM47756, MM47761, MM49721, MM47766,
MM55260, MM55266, MM55270, MM56597, M M56598, OA-7653, Orenticin, Parvodicin,
Ristocetin, Ristomycin, Synmonicin, Teicoplanin, UK-68597, UD-69542, UK-72051,
Vancomycin, and the like. The term "glycopeptide" or "glycopeptide antibiotic"
as used herein
is also intended to include the general class of glycopeptides disclosed above
on which the sugar
moiety is absent, i.e. the aglycone series of glycopeptides. For example,
removal of the
disaccharide moiety appended to the phenol on vancomycin by mild hydrolysis
gives
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vancomycin aglycone. Also included within the scope of the term "glycopeptide
antibiotics" are
synthetic derivatives of the general class of glycopeptides disclosed above,
included alkylated
and acylated derivatives. Additionally, within the scope of this term are
glycopeptides that have
been further appended with additional saccharide residues, especially
aminoglycosides, in a
manner similar to vancosamine.
101901 The term "lipidated glycopeptide" refers specifically to those
glycopeptide antibiotics
that have been synthetically modified to contain a lipid substituent. As used
herein, the term
"lipid substituent" refers to any substituent contains 5 or more carbon atoms,
preferably, 10 to 40
carbon atoms. The lipid substituent may optionally contain from 1 to 6
heteroatoms selected
from halo, oxygen, nitrogen, sulfur, and phosphorous. Lipidated glycopeptide
antibiotics are
well known in the art. See, for example, in U.S. Patent Nos. 5,840,684,
5,843,889, 5,916,873,
5,919,756, 5,952,310, 5,977,062, 5,977,063, EP 667, 353, WO 98/52589, WO
99/56760, WO
00/04044, WO 00/39156.
101911 Anti-in flarrunatory bioactive agents are also useful for dispersion
in polymer particles
used in invention compositions and methods. Depending on the intraocular site
and disease to
be treated, such anti-inflammatory bioactive agents include, e.g. analgesics
(e.g., NSAIDS and
salicyclates), steroids, antirheumatic agents, gastrointestinal agents, gout
preparations, hormones
(glucocorticoids), nasal preparations, ophthalmic preparations, otic
preparations (e.g., antibiotic
and steroid combinations), respiratory agents, find skin & mucous membrane
agents. See,
Physician's Desk Reference, 2001 Edition. Specifically, the anti-inflammatory
agent can
include dexamethasone, which is chemically designated as (119, I 61)-9-fluro-
11,17,21-
trihydroxy-16-methylpregna-1,4-diene-3,20-dione. Alternatively, the anti-
inflammatory
bioactive agent can be or include sirolimus (rapamycin), which is a triene
macrolide antibiotic
isolated from Streptomyces hygroscopicus.
101921 The polypeptide bioactive agents included in the invention
compositions and methods
can also include "peptide mimetics." Such peptide analogs, referred to herein
as "peptide
mimetics" or "peptidomimetics," are commonly used in the pharmaceutical
industry with
properties analogous to those of the template peptide (Fauchere, J. (1986)
Adv. Bioactive agent
Res., 15:29; Veber and Freidinger (1985) TINS p. 392; and Evans et al. (1987)
J. Med. Chem,
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30:1229) and are usually developed with the aid of computerized molecular
modeling. =
Generally, peptidomimetics are structurally similar to a paradigm polypeptide
(i.e., a polypeptide
that has a biochemical property or pharmacological activity), but have one or
more peptide
linkages optionally replaced by a linkage selected from the group consisting
of: - -CH2NH--,
CH2¨CH2--, --CH=CH-- (cis and trans), --COCH2--, --CH(OH)CH2--, and --CH2S0--,
by methods known in the art and further described in the following references:
Spatola, A.F. in
"Chemistry and Biochemistry of Amino Acids, Peptides, and Proteins," B.
Weinstein, eds.,
Marcel Dekker, New York, p. 267 (1983); Spatola, A.F., Vega Data (March 1983),
Vol. 1,
Issue 3, "Peptide Backbone Modifications" (general review); Morley, J.S.,
Trends. Pharm. Sci.,
(1980) pp. 463-468 (general review); Hudson, D. et al., Int. J. Pept. Prot.
Res., (1979) 14:177-
185 (--CH2NH--, CH2CH2--); Spatola, A.F. et al., Life Sci., (1986) 38:1243-
1249 (--CH2--S--);
Harm, M. M., J. Chem. Soc. Perkin Trans I (1982) 307-314 (--CH=CH--, cis and
trans);
Almquist, R.G. et al., J. Med. Chem_ (1980) 23:2533 (--COCH2--); Jennings-
Whie, C. et al.,
Tetrahedron Lettõ (1982) 23:2533 (--COCH2--); Szelke, M. et al., European
Appinõ EP 45665
(1982) CA: 97:39405 (1982) (--CH(OH)CH2--); Holladay, M. W. et al.,
Tetrahedron Lett.,
(1983) 24:4401-4404 (--C(OH)CH2--); and Hruby, V.J., Life Sci., (1982) 31:189-
199
(¨CH2¨S--). Such peptide mimetics may have significant advantages over natural
polypeptide
embodiments, including, for example: more economical production, greater
chemical stability,
enhanced pharmacological properties (half-life, absorption, potency, efficacy,
etc.), altered
specificity (e.g., a broad-spectrum of biological activities), reduced
antigenicity, and others.
1101931 Additionally, substitution of one or more amino acids within a
peptide (e.g., with a
D-Lysine in place of L-Lysine) may be used to generate more stable peptides
and peptides
resistant to endogenous peptidases. Alternatively, the synthetic polypeptides
covalently bound
to the biodegradable polymer, can also be prepared from D-amino acids,
referred to as inverso
peptides. When a peptide is assembled in the opposite direction of the native
peptide sequence,
it is referred to as a retro peptide. In general, polypeptides prepared from D-
amino acids are
very stable to enzymatic hydrolysis. Many cases have been reported of
preserved biological
activities for retro-inverso or partial retro-inverso polypeptides (US patent,
6,261,569 B1 and
references therein; B. Fromme et al, Endocrinology (2003)144:3262-3269.
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[0194] Following preparation of the polymer particle compositions loaded
with
ophthalmologic agent, the composition can be lyophilized and the dried
composition suspended
in an appropriate media prior to administration.
[0195] Any suitable and effective amount of the at least one ophthalmologic
agent can be
released with time from the polymer particles (including those in a polymer
depot formed in
vivo) or solid polymer formulation and will typically depend, e.g., on the
specific polymer, type
of particle or polymer/bioactive agent linkage, if present. Typically, up to
about 100% of the
polymer particles can be released from a polymer depot formed in vivo by
particles sized to
avoid circulation. Specifically, up to about 90%, up to 75%, up to 50%, or up
to 25% thereof
can be released from the polymer depot. Factors that typically affect the
release rate from the
polymer are the nature and amount of the polymer/ophthalmologic agent, the
types of
polymer/ophthalmologic agent linkage, and the nature and amount of additional
substances
present in the formulation.
[0196] Once the invention polymer particle delivery composition is made, as
above,
compositions are formulated for subsequent intraocular. The particle-
containing compositions
will generally include one or more "pharmaceutically acceptable excipients or
vehicles"
appropriate for implant into the interior of the eye, such as water, saline,
glycerol, polyethylene
glycol, hyaluronic acid, ethanol, etc. Additionally, auxiliary substances,
such as wetting agents,
pH buffering substances, and the like, may be present in such vehicles.
[0197] For a further discussion of appropriate vehicles to use for
particular modes of
delivery, see, e.g., Remington: The Science and Practice of Pharmacy, Mack
Publishing
Company, Easton, Pa., 19th edition, 1995. One of skill in the art can readily
determine the
proper vehicle to use for the particular bioactive agent/polymer particle
combination, size of
particle and mode of administration.
[0198] The compositions used in the invention methods optionally may
comprise an
"effective amount" of the ophthalmologic agent(s) of interest, such as an
ophthalmologic diol
incorporated into the backbone of the PEA, PEUR or PEU polymer. That is, an
amount of an
ophthalmologic agent may be included in the compositions that will cause the
subject to produce
a sufficient therapeutic or palliative response in order to prevent, reduce or
eliminate symptoms.
The exact amount necessary will vary, depending on the subject being treated;
the age and
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general condition of the subject to be treated; the severity of the condition
being treated; the
particular ophthalmologic agent selected and formulation, among other factors.
An appropriate
effective amount can be readily determined by one of skill in the art. Thus,
an "effective
amount" will fall in a relatively broad range that can be determined through
routine trials. For
example, for purposes of the present invention, an effective amount will
typically range from
about 1 pg to about 100 mg, for example from about 5 pg to about 1 mg, or
about 10 pg to about
500 pg of the active agent delivered per dose.
101991 Once formulated, the invention polymer article delivery compositions
can be
administered by implant into the interior of the eye, for example by injection
through a trochar
or needle or by insertion via a surgical incision. Dosage treatment may be a
single dose of the
invention polymer particle delivery composition, or a multiple dose schedule
as is known in the
art. The dosage regimen, at least in part, will also be determined by the need
of the subject and
be dependent on the judgment of the practitioner. Furthermore, if prevention
of disease is
desired, the polymer particle delivery composition is generally administered
prior to primary
disease manifestation, or symptoms of the disease of interest. If treatment is
desired, e.g., the
reduction of symptoms or recurrences, the polymer particle delivery
compositions are generally
administered subsequent to primary disease manifestation.
102001 The following examples are meant to illustrate, but not to limit the
invention.
EXAMPLE 1
Preparation of PEA-Ac-Bz nanoparticles and particles by the single emulsion
method
102011 PEA polymer of structure (IV) containing acetylated ends and
benzylated carboxyl
pendent groups COOCH2C6H5 (designated as PEA.Ac.Bz) (25 mg) was dissolved in 1
ml of
DCM and added to 5 ml of 0.1 % surfactant diheptanoyl-phosphatidylcholine
(DHPC) in
aqueous solution while stirring. After rotary-evaporation, a PEA.Ac.Bz
emulsion with particle
sizes ranging from 20 nm to 100 pm, was obtained. The higher the stir rate,
the smaller the sizes
of particles. Particle size is controlled by molecular weight of the polymer,
solution
concentration and equipment such as microfluidizer, ultrasound sprayer,
sonicator, and
mechanical or magnetic stirrer.
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Preparation of PEA.Ac.Bz particles containing a pain killer
[02021 PEA.Ac.Bz (25 mg) and Bupivicane (5 mg) were dissolved in 1 mL of
DCM and the
solution was added to 5 mL of 0.1 % DHPC aqueous solution while homogenizing.
Using a
rotary evaporator, a PEA.Ac.Bz emulsion with average particle size ranging
from 0.5 gm to
1000 gm, preferentially, from 1 pm to about 20 gm, have been made.
EXAMPLE 2
Preparation of polymer particles using a double emulsion method
102031 Particles were prepared using a double emulsion technique in two
steps: in the first
step, PEA.Ac.Bz (25 mg) was dissolved in 1 mL of DCM, and then 50 AL of 10 %
surfactant
diheptanoyl-phosphatidylcholine (DHPC), was added. The mixture was vortexed at
room
temperature to form a Water/Oil (W/O) primary emulsion. In the second step,
the primary
emulsion was added slowly into a 5 mL solution of 0.5 % DHPC while
homogenizing the mixed
solution. After 1 min of homogenization, the emulsion was rotary-evaporated to
remove DCM
to obtain a Water/Oil/Water double emulsion. The generated double emulsion had
suspended
polymer particles with sizes ranging from 0.5 pm to 1000 gm, with most about 1
gm to 10 gm .
Reducing such factors as the amount of surfactant, the stir speed and the
volume of water, tends
to increase the size of the particles.
EXAMPLE 3
Preparation of PEA particles encapsulating an antibody using a double emulsion
method
[02041 Particles were prepared using the double emulsion technique by two
steps: in the first
step, PEA.Ac.Bz (25 mg) was dissolved in 1 mL ofDCM, and then 50 pL of aqueous
solution
containing 60 gg of anti-Icam-1 antibody and 4.0 mg of DHPC were added. The
mixture was
vortexed at room temperature to form a Water/Oil primary emulsion. In the
second step, the
primary emulsion was added slowly into 5 nil, of 0.5 ')/o DHPC solution while
homogenizing.
After 1 min of homogenization, the emulsion was rotary-evaporated to remove
DCM to obtain
particles having a Water/Oil/VVater (W/O/W) double emulsion structure. About
75% to 98 % of
antibody was encapsulated by using this double emulsion technique.
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EXAMPLE 4
Preparation of particles having a triple emulsion structure, wherein one or
more primary
particles are encapsulated together within a polymer covering to form
secondary
microparticles.
[02051 Particles having a triple emulsion structure have been prepared by
the following two
different routes:
102061 Multi-particle Encapsulation By the first route, primary particles
were prepared using
a standard procedure for single phase, PEA.Ac.H (polymer of structure (IV)
containing
acetylated ends and free COOH pendent groups) nanoparticles were prepared to
afford a stock
sample, ranging from about 1.0 mg to about 10 mg/mL (polymer per aqueous
unit). In addition,
a solution of the PEA.Ac.Bz stock sample, with a 20% surfactant weight amount
wherein the
20% is calculated as (milligrams of surfactant)/(milligrams of PEA.Ac.Bz +
milligrams of
surfactant) was prepared. Various surfactants were explored, with the most
successful being 1,2-
Diheptanoyl-sn-glycero-3-phosphocholine. The stock sample of PEA.Ac.H
nanoparticles was
injected into a solution of PEA.Ac.Bz polymer in DCM. A typical example was as
follows:
Nanoparticle Stock Solution 100 Al
Dissolved PEA.AcBz 20 mg
CH2Cl2 2 ml
Surfactant Amount 5 mg
This first addition was referred to as the "primary emulsion." The sample was
allowed to stir by
shake plate for 5 ¨ 20 minutes. Once sufficient homogeneity was observed, the
primary
emulsion was transferred into a canonical vial that contains 0.1 % of a
surface stabilizer in
aqueous media (5-10 mL). These contents are referred to as the "external
aqueous phase".
Using a homogenizer at low speed (5000 ¨ 6000 RPM), the primary emulsion was
slowly
pipetted into the external aqueous phase, while undergoing low speed
homogenization. After 3-
5 minutes at 6000 RPM, the total sample (referred to as "the secondary
emulsion") was
concentrated in vacuo, to remove the DCM, while encapsulating the PEA.Ac.H
nanoparticles
within a continuous PEA.Ac.Bz matrix.
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102071 Preparation of Small Molecules loaded into secondary polymer
coatings. In the
second route for preparing particles having a triple emulsion structure, the
procedure described
above for making single emulsion particles was followed for the first step. In
the final step, a
polymeric coating encapsulating the single emulsion particles (i.e., the water
in oil phase) was
then prepared.
(0208] More particularly, a water in oil phase (primary emulsion) was
created. In this case a
concentrated mixture of drug (5 mg) and a surfactant (such as DHPC) was
prepared first using a
minimum volume of water. Then the concentrated Tixture was added into a DCM
solution of
PEA.Ac.Bz, and was subjected to a sonication bath for 5-10 minutes. Once
sufficient
homogeneity was observed, the contents were added into 5 ml of water while
homogenizing.
After removal of DCM by vacuum evaporation, a triple emulsion of PEA.Ac.Bz
containing
aqueous dispersion of drug was obtained.
102091 In another example, a stock sample of PEA.Ac.H nanoparticles with
drug was
prepared. PEA.Ac.H (25 mg) and drug (5 mg) were dissolved in 2 mL of DCM and
mixed with
mL of water by sonication for 5-10 minutes. Once sufficient homogeneity was
observed, the
contents were rotoevaporated to remove DCM. A typical example of preparations
made using
this method had the following contents.
PEA.Ac.H 25 mg
CH2C12 2mL
H20 5 mL
Small Molecule Drug 5 mg
The above preparation then was subjected to overnight evaporation in a Teflon
disk to further
reduce the water and yield a volume of approximately 2 mL. An exterior polymer
coating, i.e.
25 mg PEA.Ac.Bz in up to 5 mL of DCM, was combined with the primary emulsion
and the
entire secondary emulsion was stirred by vortexing for no more than I minute.
Finally, the
secondary emulsion was transferred to an aqueous media (10-15 mL) containing
0.1% surface
stabilizer, homogenized at 6000 RPM for 5 minutes, and concentrated again in
vacuo to remove
the second phase of DCM, thus yielding particles having a triple emulsion
structure.
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EXAMPLE 5
102101 Drug Capture (50%) by Triple Emulsion The following example
illustrates loading of
a small molecule drug in a polymer coating. PEA particles containing a high
loading of
bupivacaine HCI were fabricated by the triple emulsion technique, using a
minimal amount of
H20 in the primary emulsion, as compared to the double emulsion protocol
(roughly half of the
water was used). To stabilize the structure allowing for the reduction in the
aqueous phase, the
surface stabilizer that aides in solubilizing the drug in the aqueous droplets
is dissolved itself in
the internal aqueous phase before the drug is added to the internal aqueous
phase. In particular,
DHPC (amount below) was first dissolved into 100 L. H20; then 50 mg of drug
was added to
the phase. This technique allowed for loading of higher doses of drug in the
particles, with even
less water than was used in making the same sized double emulsion particles.
The following
parameters were followed during synthesis:
weight
Reagent Mg equivalence
PEA.Ac.Bz 50 50%
Bupivacaine
=
HCL 50 50%
DHPC 12.4 20% of polymer
CH2Cl2 (2 % PEA in
(solvent) 2.5 inL solvent)
H20 100 L (2:1 drug)
weight
Reagent Mg equivalence
DHPC 16 24% of polymer
2/1 ratio to
H20 5 mL solvent
EXAMPLE 6
Process for making triblock copolymer micelles with therapeutic agents
102111 First, A-B-A type triblock copolymer molecules are formed by
conjugating a chain of
hydrophobic PEA or PEUR polymer at the center with water soluble polymer
chains containing
alternating units of PEG and at least one ionizable amino acid, such as lysine
or glutamate, at
both ends. The triblock copolymer is then purified.
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102121 Then micelles are made using the triblock copolymer. The triblock
copolymer and at
least one bioactive agent, such as a small molecule drug, a protein, peptide,
a lipid, a sugar,
DNA cDNA or RNA, are dissolved in aqueous solution, preferably in a saline
aqueous solution
whose pH has been adjusted to a value chosen in such a way that at least a
portion of the
ionizable amino acids in the water soluble chains is in ionized form to
produce a dispersion of
the triblock polymer in aqueous solution. Surface stabilizer, such as
surfactant or lipid, is added
to the dispersion to separate and stabilize particles to be formed. The mixed
solution is then
stirred with a mechanical or magnetic stirrer, or sonicator. Micelles will be
formed in this way,
with water-soluble sections mainly on the shell, and hydrophobic sections in
the core,
maintaining the integrity of micellar particles. The micelles have high
porosity for loading of
the active agents. Protein and other biologics can be attracted to the charged
areas in the water-
soluble sections. Micellar particles formed are in the size range from about
20nm to about 200
nm.
EXAMPLE 7
Polymer coating on particles made of different polymer mixed with drug
102131 Use of single emulsion leaves the problem that, although particles
can be made very
small (from 20 nm to 200 nm), the drug is matrixed in the particles and may
elute too quickly.
For double and triple emulsion particles, the particles are larger than is
prepared by the single
emulsion technique due to the aqueous solution inside. However, if the same
polymer is used
for coating the particles as is used to matrix the drug, the solvent used in
making the third
emulsion (the polymer coating) will dissolve the matrixed particles, and the
coating will become
part of the matrix (with drugs in it). To solve this problem, a different
polymer than is used to
matrix the drug is used to make the coating of the particles and the solvent
used in making the
polymer coating is selected to be one in which the matrix polymer will not
dissolve.
102141 For example, PEA can be dissolved in ethanol but PLA cannot.
Therefore, PEA can
be used to matrix the drug and PLA can be used as the coating polymer, or vice
versa. In
another example, ethanol can dissolve PEA but not PEUR and acetone can
dissolve PEUR but
cannot dissolve PEA. Therefore, PEUR can be used to matrix the drug and PEA
can be used as
the coating polymer, or vice versa.
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102151 Therefore, the general process to be used is as follows. Using
polymer A, prepare
particles in solution (aqueous if polymer A is PEA or PEUR) using a single
emulsion procedure
to matrix drug or other bioactive agent in the polymer particles. Dry out the
solvent by
lyophilization to obtain dry particles. Disperse the dry particles into a
solution of polymer B in a
solvent that does not dissolve the polymer A particles. Emulsify the mixture
in aqueous
solution. The resulting particles will be nanoparticles with a coating of
polymer B on particles
of polymer A, which contain matrixed drug.
EXAMPLE 8
102161 In this example a PEA polymer containing a residue of P-Estradiol in
the main PEA
polymer backbone was prepared.
102171 Materials 17-13-estradiol (estra-1,3,5(10)-triene-3,1713-diol), L-
lysine, benzyl alcohol,
sebacoyl chloride, 1,6-Hexanediol, p-nitrophenol, triethylamine, 4-N,N-
(dimethylamino)pyridine (DMAP), N,N'-dicyclohexylcarbodiimide (DCC), anhydrous
N,N-
dimethylformamide (DMF), anhydrous dichloromethane (DCM), trifluoroacetic acid
(TFA) , p-
toluenesulfonic acid monohydrate (Aldrich Chemical Co., Milwaukee, WI),
anhydrous toluene,
Boc-L-leucine monohydrate (Calbiochem-Novabiochem, San Diego, CA) were used
without
further purification. Other solvents, ether and ethyl acetate (Fisher
Chemical, Pittsburgh, PA).
102181 Synthesis of Monomers and Polymers Synthesis of bioactive PEAs
involved three
basic steps: (1) synthesis of bis-electrophiles: di(p-nitrophenyl) esters of
dicarboxylic acid (here
of sebacic acid, compound 1); (2) synthesis of bis-nucleophiles: di-p-
toluenesulfonic acid salts
(or di-TFA salt) of bis(L-leucine)-diol-diesters (compounds 3 and 5) and of L-
lysine benzyl ester
(compound 2); and (3) solution polycondensation of the monomers obtained in
steps (1) and (2).
102191 Synthesis of di-p-nitrophenyl esters of sebacic acid (compound 1) Di-
p-
nitrophenyl ester of sebacic acid was prepared by reaction of sebacoyl
chloride with p-
nitrophenol as described previously (Katsarava et al. J. Polym. Sci. Part A:
Polym. Chem. (1999)
37. 391-407) (scheme 4):
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9 0 0
Cl-c-(cH2)8-c-ci f 02N =OH __________ - Triethylamine 02N 0-
6-(CH2)8-9-0 11 NO2
0
( 1 )
Scheme 4
A di-p-toluenesulfonic acid salt of L-lysine benzyl ester was prepared as
described earlier (US
6,503,538) by refluxing of benzyl alcohol, toluenesulfonic acid monohydrate
and L-lysine
monohydrochloride in toluene, while applying azeotropic removal of generated
water (scheme
Scheme 5).
_..TTooisuoe
HCI.H2N-(CH2)4-CH-NH2 + HOCH2-C6H5 Tos0H.H2N-(CH2)4-CH-NH2.Tos0H
COOH reflux,
CO2CH2C6H5
-H20
( 2 )
Scheme 5
102201 Synthesis of acid salts of bis(a-amino acid) diesters (3), (5) Di-p-
toluenesulfonic
acid salt of bis(L-leucine) hexane-1,6-diester (compound 3) was prepared by
modified procedure
of the previously published method as shown in scheme 3.
10221) L-Leucine (0.132 mol), p-toluenesulfonic acid monohydrate (0.132
mol) and
1,6-hexanediol (0.06 mol) in 250 mL of toluene were placed in a flask equipped
with a Dean-
Stark apparatus and overhead stirrer. The heterogeneous reaction mixture was
heated to reflux
for about 12 h until 4.3 mL (0.24 mol) of water evolved. The reaction mixture
was then cooled
to room temperature, filtered, washed with acetone, and recrystallized twice
from
methanol/toluene 2:1 mixture. Yields and Mp were identical to published data
(Katsarava et al.,
supra) (see scheme 6).
Tos0H H H
H2N-CH-G-OH + HO-(CH2)6-0H
HOTosH2N-C-C-0-(CH2)6-0-6-9--NH2.Tos0H
CH2 Toluene,
reflux CH2 CH2
CH(CH3)2 CH(CH3)2 CH(CH3)2
( 3 )
Scheme 6
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A di-TFA salt of bis-(L-leucine)- estradioI-3,17 O-diester (compound 5) was
prepared by a two
step reaction. 17P-Estradiol was first reacted with boc-protected L-leucine,
applying
carbodiimide mediated esterification, to form compound 4. In a second step,
boc groups were
deprotected using TFA, converting at the same time into a di-TFA salt of di-
amino monomer
(compound 5) (see scheme 7).
0
OH
ON)LO+
DCC, DMAP 0 H
2 Boc = DMF, RT
COOH opop
74%
HO
H
TFA.H2N\
(4) TFA \43 0 NH2.TFA
DCM
= 0 (5)
Scheme 7
102221 Preparation of Bis(Boc-L-leucine)estradio1-3,171S-diester (4) 1.5 g
(5.51 mmol) of
17P¨estradiol, 3.43 g (13.77 mmol) Boc-L-leucine monohydrate and 0.055 g (0.28
mmol) of
p-toluenesulfonic acid monohydrate were dissolved into 20 mL of dry N,N-
dimethylformamide
at room temperature under a dry nitrogen atmosphere. To this solution lOg of
molecular sieves
were added and stirring continued for 24 h. Then, 0.067 g of DMAP and 5.4g of
(26.17 mmol)
DCC were introduced into the reaction solution and stirring was continued.
After 6 h (no
discoloration of the reaction was observed), 1 mL of acetic acid was added to
destroy the excess
of DCC. Precipitated urea and sieves then were filtered off and filtrate
poured in 80 mL of
water. Product was extracted three times with 30 mL of ethylacetate, dried
over sodium sulfate,
solvent evaporated, and the product was subjected to chromatography on a
column (7:3 hexaries:
ethylacetate). A colorless glassy solid of pure compound 4 obtained in a 2.85
g, 74 % yield and
100% purity (TLC) and was further converted to compound 5.
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102231 Di-TFA salt of bis(L-leucine)estradio1-3,1713-diester (compound 5).
Deprotection
of Boc-protected monomer (compound 4) was carried out substantially
quantitatively in10 mL of
dry dichloromethane, by adding 4 mL of dry TFA. After 2 h of stirring at room
temperature, a
homogenous solution was diluted with 300 mL of anhydrous ether and left in a
cold room over
night. Precipitated white crystals were collected, washed twice with ether,
and dried in a
vacuum oven at 45 C. Yield 2.67 g (90%). Mp = 187.5 C.
10224] Polymer Synthesis. Synthesis of therapeutic PEA was carried out in
DMF in mild
conditions (60 C): 4 eq. activated di-acid monomer (compound 1) was reacted
with
combinations of the di-amino monomers 1.5 eq. (compound 2), 1.5 eq. (compound
5) and 1 eq.
of (compound 3).
[02251 Triethylamine 1.46 mL (10.47 mmol) was added at once to the mixture
of monomers
(compound l)(4.986 mmol), (compound 2) (1.246 mmol), (compound 3) (1.869
mmol),
(compound 5) (1.869 mmol) in 3 mL of dry DMF and the solution was heated to 60
C while
stirring. The reaction vial was kept at the same temperature for 16h. A yellow
viscous solution
was formed then was cooled down to room temperature, diluted with 9 mL of dry
DMF, added
0.2 mL of acetic anhydride, and after 3 h precipitated out three times: first
in water, then from
ethanol solution into ethylacetate and lastly, from chloroform in ethyl
acetate. A colorless
hydrophobic polymer was cast as a film from chloroform : ethanol (1:1) mixture
and dried in
vacuum. Yield: 1.74g (70%).
102261 Materials Characterization The chemical structure of monomers and
polymer were
characterized by standard chemical methods. NMR spectra were recorded by a
Bruker AMX-
500 spectrometer (Numega R. Labs Inc. San Diego, CA) operating at 500 MHz for
11-1 NMR
spectroscopy. Deuterated solvents CDCI3 or DMSO-d6 (Cambridge Isotope
Laboratories, Inc.,
Andover, MA) were used with tetramethylsilane (TMS) as internal standard.
102271 Melting points of synthesized monomers were determined On an
automatic Mettler-
Toledo FP62 Melting Point Apparatus (Columbus, OH). Thermal properties of
synthesized
monomers and polymers were characterized on Mettler-Toledo DSC 822e
differential scanning
calorimeter. Samples were placed in aluminum pans. Measurements were carried
out at a
scanning rate of 10 C/min under nitrogen flow.
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1022811 The number and weight average molecular weights (Mw and Mn) and
molecular
weight distribution of synthesized polymer was determined by Model 515 gel
permeation
chromatography (Waters Associates Inc. Milford, MA) equipped with a high
pressure liquid
chromatographic pump, a Waters 2414 refractory index detector. 0.1% of LiC1
solution in N,N-
dimethylacetamide (DMAc) was used as eluent (1.0 mL/min). Two Styragel HR 5E
DMF
type columns (Waters) were connected and calibrated with polystyrene
standards.
[0229] Tensile Properties: tensile strength, elongation at break and
Young's Modulus were
measured on a tensile strength instrument (Chatillon TCD200, integrated with a
PC (NexygenTM
FM software)(Chatillon, Largo, FL) at a crosshead speed of 100 mm/min. The
load capacity
was 50 lbs. The film (4 x 1.6 cm) had a dumbbell shape and thickness of about.
0.125 mm.
102301 Results: Four different monomers were copolymerized by
polycondensation of
activated monomers, affording copoly PEA containing 17 % w/w steroid-diol load
on a total
polymer weight basis. Chemical structure of the product therapeutic polymer
composition,
containing fragments of 170-estradiol, L-Leucine, L-Lysine (013n), 1,6-
hexanediol and sebacic
acid is depicted in Formula (XIX).
0
¨ CH3 9 0
0
____ HN 9H-C" -0 \ wip 0-C-CH-NH-C-(CH2)8-6¨
,H2 sH2
CH(CH3)2 CH(CH3)2
_ iSn/4
o 9
¨8-(cH2),-c-HN-HC-(cH04-NH ___
gIcH08-2 HN-HC-?-0 (CH2)6-0-g-CH NH __________________________________
CO2CH2C6H5 ni4 012
CH(CH3)2 CH(CH3)2 1511/4
Formula (XIX) =
Three monomers: bis-p-toluenesulfonic acid salts of L-lysine-benzyl ester
(compound 2), bis(L-
leucine) 1,6-hexane diester (compound 3), and bis(p-nitrophenyl) sebacate
(compound 1) were
prepared according to the literature and characterized by melting point and
proton NMR
spectroscopy. Results were in agreement with those reported in literature.
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10231 In this example a PEA polymer containing a residue of 1713-Estradiol
in the main
polymer backbone was prepared, where both hydroxyls of the diol steroid were
incorporated into
monomer via ester bonds using a carbodiimide technique. The final monomer
introduced into
the polymerization reaction was a TFA salt. After polycondensation, a high
molecular weight
copolymer was obtained. Gel permeation chromatography yielded an estimated
(PS) weight
average Mw = 82,000 and polydispersity PDI = 1.54. The product copolymer was
partially
soluble in ethanol (when dry), well soluble in chloroform, chloroform:ethanol
1:1 mixture,
dichloromethane, and in polar aprotic organic solvents: DMF, DMSO, DMAc.
102321 Glass transition temperature was detected at Tg = 41 (midpoint,
taken from the
second heating curve) and a sharp melting endotherm was detected at 220 C by
Differential
scanning calorimetry (DSC) analysis. This result leads to the conclusion that
the polymer has
semi-crystalline properties.
[02331 The therapeutic polymer formed a tough film when cast from
chloroform solution.
Tensile characterization yielded the following results: Stress at break 28.1
MPa, Elongation
173%, Young's Modulus 715 MPa.
EXAMPLE 9
102341 This Example illustrates synthesis of a therapeutic PEUR polymer
composition
(Formula V) containing a therapeutic diol in the polymer backbone is
illustrated in this example.
A first monomer used in the synthesis is a di-carbonate of a therapeutic diol
with a general
9 9
chemical structure illustrated by formula R5-0-C-0-R6-0-C-0-R5 is formed using
a known
procedure (compound (X) as described in U.S. Patent 6,503,538) wherein R5 is
independently
(C6-Cio) aryl (e.g. p-nitrophenol, in this example), optionally substituted
with one or more nitro,
cyano, halo, trifluoromethyl or trifluoromethoxy; and at least some of p-
nitrophenol. At least
some of R6 is a residue of a therapeutic diol as described herein, depending
upon the desired
drug load. In the case where all of R6 is not the residue of a therapeutic
diol, each diol would
first be prepared and purified as a separate monomer. For example, di-p-
nitropheny1-3,17b-
estradiol-dicarbonate (compound 6) can be prepared by the method of Scheme 8
below:
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OH 0
0
0 1, 0 lik
2 02N . 0-1I-01 + Mlle Base 02N m
AD o-u-o O-ILO NO2
411 =
HO IPP ( 6 )
Scheme 8
Polycondensation of compound X from U.S. Pat. 6,503,538 (in our example
compound 6) with
the monomers described above yields an estradiol-based co-poly(ester urethane)
PEUR
(compound 11):
HOTos.H2N ANH2.Tos0H TFA.H 2141\4
) 0 .... 0 NH2.TFA
0 0 0 0
( 2 )
( 5 )
0
0
02N 410 0-U-0 1, 111
0 = 0-1I-0 It NO2
( 6 )
wherein the reaction scheme is as follows
TEA
3eq. (compound 5) + 1 eg. (compound 2) + 4eg. (compound 6) ----4 (compound 11
)
DMF
19
C-0 ek 40C H3 0 -
0
g-NH-CH- C-0 lik Anik CH3 0
0 = CH2 wo O--CH-NH _______
CH2
CH(CH3)2 CH(CH3)2_ 3ni4
,
H 0 H3C
-{ Aim 0
HNICH2)4-C-HN-8-0 *W 0-8 ,-
CO2CH2C8H5 O
Compound (11)
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EXAMPLE 10
Monomer synthesis for preparation of PEU polymers
102351 Preparation of diamine type monomers--di-p-toluenesulfonic acid salt
of L-lysine
benzyl ester (L-Lys(0Bn), Compound 2) and di¨toluenesulfonic acid salt of
bis(L-leucine)-
hexane-1,6-diester, (compound 3)--were described in previous Example 8.
102361 Preparation of Di-p-toluenesulfonic acid salt of bis(L-leucine)-
1,4:3,6-
dianhydrosorbitol-diester (Compound 7) was conducted as described previously
(Z.Gomurashvili et al. J. Macromol. Sci. ¨Pure. Appl. Chem. (2000) A37: 215-
227).
H9
OH
HOTos.H2N¨c¨C¨Ow... 0-8-c, ¨NH2.Tos0H
CH2
0 9E12
CH(CH3)2 CH(C1-13)2
Compound 7
wherein L-leucine (0.132 mol), p-toluenesulfonic acid monohydrate (0.135 mol)
and isosorbide
(0.06 mol) in 250 mL of toluene were placed in a flask equipped with a Dean-
Stark apparatus
and overhead stirrer. The heterogeneous reaction mixture was heated to reflux
for about. 12 h
until 4.3 mL (0.24 mol) of water evolved. The reaction mixture was then cooled
to room
temperature, filtered, washed with acetone and recrystallized twice from
methanol/toluene 2:1
mix. Yields and Mp were identical to published data (Z.Gomurashvili et
al.supra).
EXAMPLE 11
Preparation of PEU 1-L-Leu-6 (Polymer entry # 2, Table 2)
102371 To a suspension of 6.89 g (10 mmol) of di-p-toluenesulfonic acid
salt of bis(L-
leucine)-10-hexanediol-diester in 150 mL of water, 4,24 g (40 mmol) of
anhydrous sodium
carbonate was added, stirred at room temperature for 30 min. and cooled to 2
C to 0 C. In
parallel, a solution of 0.9893 g (10 mmol) of phosgene in 35 mL of chloroform
was cooled to 10
tol 5 C. The first solution was placed into a reactor for interfacial
polycondensation and the
second solution was quickly added in bolus and stirred briskly for 15 min.
Then the chloroform
layer was separated, dried, over anhydrous Na2SO4, and filtered. The obtained
solution was
evaporated and the polymer yield was dried in vacuum at 45 C. Yield was 82%.
For II-1 and "C
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NMR see Fig 2 and Fig 3. Elemental analysis: for C19H34N205, calculated
values: C: 61.60%, H:
9.25%, N: 7.56%; Found values: C: 61.63%, H: 8.90%, N: 7.60.
EXAMPLE 12
Preparation of PEU 1-L-Leu-DAS (polymer: entry # 5, Table 2)
HO
S 9 k, 9
H cH2 C
CH(CH3)2 cH2
CH(CH3)2 n
Compound 15
(02381 A cooled solution (ice-bath) of 5 g (6.975 mmole) of bis (L-leucine)-
1,4:3,6-
dianhydrosorbitol-diester (compound 7) and 2.4 g of sodium carbonate in 40 mL
of water was
prepared. To the cooled solution, 70 mL of chloroform was added with vigorous
stirring and
then 3.7 mL of 20% phosgene solution in toluene (Fluka) was introduced.
Poly(ester urea)
formed rapidly with evolution of heat. After the reaction had been stirred for
10 min, the
organic layer was rotoevaporated and residual polymer was filtered, washed
several times with
water, and dried in vacuum over night. Yield of product was 1.6 g. (57%).
Polymer properties
are as summarized in Table 2.
EXAMPLE 13
102391 This example describes a degradation study conducted to compare
degradation rates
over time of a PEU polymer 1-L-Leu-4. Circular PEU films of 4 cm diameter and
400-500 mg
each, were placed into the glass beakers containing 10 ml of 0.2 M phosphate
buffer solution of
pH 7.4 with 4 mg of an enzyme, either ct-chymotrypsin or lipase, or without
enzymes. The glass
vessels were maintained at 37 C. Films were removed from the enzyme solution
after
predetermined time, dried up to constant weights, and weighed. Then the films
were placed into
the fresh solution of either enzyme or pure buffer and all the procedures
described above were
repeated. Weight changes per unit surface area of the sample were calculated
and represented
graphically vs. biodegradation time. The results of the study showed that the
PEU polymer has a
degradation profile that is almost zero order, corresponding to a surface
degradation profile.
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[0240] The invention has been described with reference to various
specific and
preferred embodiments and techniques. However, it should be understood that
many
variations and modifications might be made while remaining within the scope of
the
invention.
[0241] Although the invention has been described with reference to the
above
examples, it will be understood that modifications and variations are
encompassed within the
scope of the invention. Accordingly, the invention is limited only by the
following claims.
